A conserved, N-terminal tyrosine signal directs Ras for inhibition by Rabex-5.

Dysregulation of the Ras oncogene in development causes developmental disorders, "Rasopathies," whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.

Introduction

In Drosophila, Ras proteins are represented by Ras1/Ras85D and Ras2/Ras64B. Drosophila Ras1/Ras85D corresponds to mammalian H-Ras, N-Ras, and K-Ras and is distinct from the closely related Ras2/Ras64B protein represented by R-Ras in mammals. Using the convention that predominates in the literature, here we refer to Drosophila Ras1/Ras85D as Ras; we refer collectively to mammalian H-Ras, N-Ras, and K-Ras as Ras or individually to specific isoforms as H-Ras, N-Ras, and K-Ras as appropriate; and we refer to Ras2/Ras64B as Ras2 for the Drosophila protein and R-Ras for the mammalian protein. Drosophila Ras and mammalian H-Ras N-Ras and K-Ras share sequence identity in their N-termini but diverge in a C-terminal region called the HyperVariable Region or HVR [1; for review, 2–3] (depicted schematically in S1 Fig). It is well accepted that one of the roles of the HVR is to direct the membrane association of Ras in part by the C-terminal CAAX box (cysteine, aliphatic amino acid, aliphatic amino acid, any amino acid) [1; for review, 2–3].

Ras signaling regulates cell proliferation, growth, differentiation, and cell survival by signaling to a range of downstream effectors including Raf/ERK/MAPK, RalGDS, and PI3K among others [1-12]. Consequently, Ras dysregulation in development alters patterning and causes developmental disorders collectively called “Rasopathies” [for review, 4–9]. Mutational activation and amplification of Ras in differentiated tissues are implicated in cancer [for review, 10–12]. Therefore, mechanisms of attenuating Ras activity are crucial for proper development and to prevent disease.

Rabex-5 (also called RabGEF1), an A20-like E3 ubiquitin ligase, promotes inhibitory mono- and di-ubiquitination of Drosophila Ras and mammalian H-Ras and N-Ras to restrict signaling to downstream effectors [13-16]. Rabex-5 inhibits both wild-type Ras and also the constitutively active oncogenic mutant RasG12V (also referred to as RasV12 in the literature) [13-16].

No signal in Ras has been reported to direct its inhibition by Rabex-5, and no ubiquitination targeting motif has been ascribed to Rabex-5 or the A20 family of ubiquitin ligases. We mapped a ubiquitination signal in Drosophila Ras; we report here that Rabex-5 inhibition of Drosophila Ras requires Ras N-terminal tyrosine 4 (Y4). Phenylalanine substitution mutants of Ras at Y4 (to prevent phosphorylation) were insensitive to Rabex-5-mediated ubiquitination in S2 cells and showed Ras gain-of-function phenotypes in vivo. Glutamic acid substitution mutants of Ras at Y4 (to mimic the charge of phosphorylation) showed increased Rabex-5-mediated ubiquitination in S2 cells, and glutamic acid substitution mutants of RasG12V at Y4 suppressed oncogenic Ras phenotypes in vivo, dependent on the presence of Rabex-5. JAK2 and SRC kinases are capable of promoting phosphorylation of RasWT at Y4, whereas JAK2, SRC, and EGFR can promote phosphorylation of an oncogenic form of Ras, RasG12V, at Y4 as measured by recognition by anti-pY4 antibodies.

Results and discussion

An N-terminal tyrosine-based signal directs Ras for mono- and di-ubiquitination

To elucidate the molecular mechanism of Ras inhibition by Rabex-5 and to advance our understanding of the A20 family of E3 ubiquitin ligases, we mapped a signal in Drosophila Ras responsible for Rabex-5 mediated ubiquitination with a deletion strategy (S1A Fig). In our previous work, we used a double FLAG-His6 tag on full length Ras [13, 16]. Deletion constructs were tagged with a triple tag of GFP-FLAG-His6 so that smaller constructs (corresponding to larger deletions) would be large enough to eliminate concerns of peptide instability. Ubiquitin conjugates of deletion constructs were isolated from Schneider S2 cells using nickel purification (to isolate the His6 tag). Visualization of the conjugates was achieved using the FLAG tag (to visualize Ras) and HA (to visualize ubiquitin which was expressed from an HA-Ub plasmid) as done previously [13, 16]. Ras is also regulated by the E3s Nedd4 [17], βTRCP [18], and LZTR1 [19-20]. Therefore, to map a Rabex-5 ubiquitination signal but without excluding Nedd4, βTRCP, or LZTR1 signals, our initial deletion strategy followed ubiquitination of Ras in Schneider S2 cells without Rabex-5 supplementation (S1A–S1D Fig). As noted, Ras membrane association is directed by a C-terminal CAAX signal which is represented by the amino acids CKML in Drosophila Ras. To properly localize N-terminal constructs, we tagged each deletion construct at its C-terminus with the Drosophila Ras CAAX box CKML (depicted schematically in S1A Fig; sequences listed in the methods section).

Previous work by Jura et al. reported the importance of the HVR for Ras ubiquitination. Inhibitory ubiquitination of H-Ras and N-Ras but not K-Ras was reported in mammalian cells [15]. Notably, replacing the K-Ras HVR with the H-Ras HVR conferred ubiquitination onto K-Ras [15]. This could have reflected the requirement for specific sequences in the H-Ras HVR not present in the K-Ras HVR; alternatively, this could have reflected the importance of the HVR in directing the localization of each Ras isoform to a compartment where the ubiquitination occurs. We report here that the Drosophila Ras HVR was neither sufficient nor required for Ras ubiquitination. C-terminal constructs were not ubiquitinated (S1A and S1B Fig). After narrowing the region sufficient for ubiquitination to the N-terminal 20 amino acids of Ras, we tested the ability of the N-terminal 20 amino acids to serve as a competitive inhibitor. Expressing GFP-Myc-tagged 1-20CKML peptides in excess prevented the formation of Ras-ubiquitin conjugates of full-length FLAG-His6 tagged RasWT isolated on nickel beads and detected by anti-HA antibodies (S1E Fig), whereas GFP-myc tagged peptides of a different 20 amino acid region in excess had no effect on RasWT ubiquitin conjugates (S1E Fig). We further narrowed the region sufficient to confer Ras ubiquitination in S2 cells to the N-terminal 10 amino acids of Ras (Fig 1A, S1A Fig, S1F and S1G Fig). Co-transfecting cells with Rabex-5 increased the ubiquitination of this region (Fig 1A, S1F and S1G Fig) but not of other small regions of Ras (S1F Fig).

Fig 1

Ras Tyrosine 4 is required for Rabex-5-mediated Ras ubiquitination.

(A) Flag-His6-GFP tagged Ras (RasWT) or FLAG-His6-GFP tagged Ras N-terminal fragments tagged with C-terminal localization signal CKML (1–10 CKML, 1–20 CKML) were co-transfected into Schneider S2 cells with HA-Ub with or without Rabex-5 and purified on nickel beads as done previously [13, 16]. The N-terminal 10 amino acids of Ras contain a signal sufficient to confer ubiquitination onto GFP in the pattern of full length Ras and to support Rabex-5-mediated ubiquitination (image of entire gel in S1G Fig). The bands recognized by both anti-FLAG (the tag on Ras) and anti-HA (the tag on ubiquitin) antibodies represent ubiquitinated species of Ras and are marked by an asterisk, *. Other bands in the anti-HA gel reflect non-Ras, co-purifying ubiquitinated proteins. (B-D) Localization in S2 cells of FLAG-His6 tagged RasWT (B), RasY4E (C), and RasY4F (D) visualized by staining for FLAG. Boxes represent 20 μm square regions. (E) Western blot of FLAG-His6 RasWT and Y4 mutants purified from S2 cells on nickel beads. Ubiquitin conjugates (anti-HA antibodies, upper blot) and total Ras (anti-FLAG antibodies, lower blot) show an increase in basal ubiquitination for phoshomimic Ras, RasY4E, compared to RasWT (lane 3 compared to lane 1) and Rabex-5-mediated ubiquitination (increase in lane 4 compared to lane 3 versus the increase in lane 2 compared to lane 1). Non-phosphorylatable Ras, RasY4F, shows decreased basal ubiquitination (lane 5 compared to lane 1) and less responsiveness to Rabex-5 (lane 6 compared to lane 5). Quantification of these experiments shows the percent of Ras conjugated to ubiquitin (graph in E’) and the relative Rabex-5 mediated ubiquitination (graph in E”). *** indicates p<0.0001 from CHITEST function in Excel for Chi-square statistical analysis comparing the percentage of Ras construct in unconjugated or ubiquitin conjugated species between indicated samples. Western Analysis utilized the Licor Odyssey. Gels in this figure showed non-saturating band intensities; quantification of bands in E’ and E” utilized non-saturating signals in the linear range.

Because the HVR was neither sufficient for ubiquitination nor required for ubiquitination of constructs, our results taken together with the previous work of Jura et al.[15], could be consistent with a requirement for the HVR to direct Ras localization [1; for review, 2–3] to a specific compartment where a sequence shared by H-Ras and K-Ras would then be recognized. In fact, the N-termini of Drosophila Ras and H-Ras, N-Ras, and K-Ras are identical (alignment, S2C Fig). While we cannot rule out a contribution from amino acids 11–189, the first 10 amino acids of Ras were sufficient to confer ubiquitination.

Normally, RasWT and RasG12V proteins associate with the plasma membrane and are also found in the cytoplasm [1–3, 15] (Fig 1B, S1H Fig S2E–S2E” Fig). Previous work by the Bar-Sagi group has shown that a non-ubiquitinated mutant of mammalian H-Ras (in which all solvent exposed lysines were mutated to arginine) had increased localization to the Golgi from which Ras proteins are known to actively signal [15]. In contrast, an H-Ras ubiquitin fusion protein showed decreased Golgi localization and increased localization in an early endosomal compartment [15]. This suggests that the mechanism of inhibitory ubiquitination is to sequester ubiquitinated Ras proteins away from downstream effectors they would encounter at the membrane or in the Golgi by retaining it in the early endosome. We over-expressed Rabex-5DPYT (which maintains E3 activity but has impaired Rab5 GEF function) to increase the inhibitory ubiquitination of endogenous Ras proteins without affecting the endosomal compartment in the developing eye using GMR-gal4. Staining for endogenous Ras using anti-pan-Ras antibodies at a specific time window 48 hours after puparium formation revealed a dramatic redistribution of Ras to an intracellular compartment (S1I Fig) not seen in controls (S1H Fig). Taken together with work from the Bar-Sagi group showing redistribution of a Ras ubiquitin fusion construct [15], we speculate that Rabex-5 activity can promote Ras re-localization to prevent its signaling to downstream effectors in a highly conserved fashion.

Most cellular Ras is GDP-loaded [21]. Mutation at or close to Ras codon 12 biases Ras to a GTP-loaded conformation [22]. To define a signal in both GDP- and GTP-loaded Ras conformations, we created alanine substitution mutants of the first ten amino acids in FLAG-His6 tagged full-length Drosophila RasWT and FLAG-His6 tagged full-length Drosophila RasG12V. Y4A and V7A substitution reproducibly decreased ubiquitination in both RasWT and RasG12V (S2A and S2B Fig). Curiously, we saw differences in alanine scanning between Ras and RasG12V constructs (S2A and S2A’ Fig, summarized in S2B Fig). E3A and K5A substitutions decreased ubiquitination of only RasG12V (S2A’ Fig, S2B Fig).

The tyrosine and valine important for ubiquitination of Drosophila RasWT and RasG12V and the lysine important for ubiquitination in RasG12V are entirely conserved in human H-Ras, N-Ras, and K-Ras as well as in Drosophila Ras2 (S2C Fig). To establish if Rabex-5 can also promote ubiquitination of Ras2, we expressed FLAG-His6 tagged Ras2 in Schneider S2 cells. Rabex-5 promoted Ras2 ubiquitination (S2D Fig) to a similar extent as RasWT. The crucial tyrosine is not conserved in R-Ras (alignment, S2C Fig), so it is unclear if the ability of Rabex-5 to promote Ras2 ubiquitination would be conserved for human R-Ras.

In transfected Schneider S2 cells, most cells show Ras associated with the membrane and also in intracellular puncta in the cytoplasm. In a population of transfected cells, some individual cells showed enrichment of Ras signal at the membrane or in intracellular puncta (S2E–S2E” Fig). Because work by the Bar-Sagi group indicated the importance of localization for Ras ubiquitination [15], we assessed localization of the alanine scanning constructs. Localization of each alanine mutant disrupting ubiquitination confirmed that a lack of ubiquitination did not result from a failure to localize to the plasma membrane and these cytoplasmic puncta (S2F–S2M Fig).

Previous proteomic studies have identified phosphorylation in mammalian Ras at Y4 [23] and also at other tyrosines [24-30]. RasWT purified from S2 cells was recognized by anti-phosphotyrosine (p-tyr) antibodies (S2N–S2P Fig). RasY4F (phenylalanine substitution to preserve structure but without the hydroxyl group) showed decreased recognition by anti-p-tyr antibodies compared to RasWT (S2O and S2P Fig) consistent with phosphorylation at Y4 and also at other tyrosines in Drosophila Ras as seen in mammalian Ras.

Negative charge at Y4 increases Rabex-5 mediated Ras ubiquitination

We created glutamic acid substitution mutant RasY4E to mimic the charge of phosphorylation in order to test the effects of negative charge at Y4 in vitro and in vivo. RasY4E and RasY4F associated with the plasma membrane and intracellular puncta as seen with RasWT (Fig 1B–1D). The basal level of RasY4E ubiquitination increased compared to RasWT, and Rabex-5-mediated RasY4E ubiquitination increased compared to Rabex-5-mediated ubiquitination of RasWT (Fig 1E, quantified in Fig 1E’ and 1E”, raw data in S1 File). In contrast, RasY4F showed lower basal ubiquitination and no Rabex-5-mediated ubiquitination (Fig 1E–1E”).

RasY4F shows Ras gain-of-function phenotypes in vivo

To investigate a biological role for Drosophila Ras Y4, we created inducible transgenic lines for RasWT, RasY4E, RasY4F, RasG12V, and double mutants RasY4E,G12V and RasY4F,G12V. Each transgene was tagged with the N-terminal FLAG-His6 tag used in our in vitro studies and was inserted at the same attp40 genomic site to rule out position insertion effects. Transgenes expressed at similar levels (S3B Fig for larvae). RasY4E and RasY4F expressed with Act5C-gal4 rescued the early lethality of Ras loss-of-function alleles to the same extent as RasWT transgenes (S3I Fig), suggesting that genetically Y4E and Y4F mutations are capable of carrying out Ras function and do not inactivate Ras inherently.

When Ras signaling is kept within an appropriate range during development, growth, proliferation, and cell fate decisions occur normally. When Ras signaling is in excess, a variety of growth and patterning phenotypes result depending on the developmental timing and tissue context. Expression of RasWT in many contexts such as in the eye (using GMR-gal4 and ey-gal4) and the wing (using c765-gal4) does not disrupt growth or disrupt patterning and cell fate decisions (summarized in S3A Fig), presumably because endogenous mechanisms of regulating Ras buffer the increased Ras expression to within the normal range. However, expressing RasWT with constitutive driver Tub-gal4 or dorsal wing driver MS1096-gal4 resulted in the Ras gain-of-function phenotype of ectopic wing veins (Fig 2B, 2B’ and 2E; S3D and S3D’ Fig, S3G Fig) compared to control wings (Fig 2A, 2A’ and 2D; S3C and S3C’ Fig, S3F Fig). RasY4F increased wing vein disruption (Fig 2C, 2C’ and 2F; S3E and S3E’ Fig, S3H Fig) compared to RasWT and statistically significantly decreased wing size compared to controls (Fig 2G, raw data in S2 File). The increase in severity of phenotype suggests that Y4F substitution increases Ras activity.

Fig 2

Non-phosphorylatable Ras shows Ras gain-of-function phenotypes in vivo.

(A) Control wing (Tub-gal4/+). (B) RasWT expression driven by Tub-gal4. Ectopic longitudinal vein material is seen anterior to the L2 longitudinal vein (arrow, enlarged in B’) and on the posterior crossvein (arrow). (C) RasY4F expression driven by Tub-gal4. Ectopic longitudinal vein material is seen anterior and posterior to the L2 longitudinal vein (arrows, enlarged in C’). The ectopic wing vein phenotype is enhanced upon Y4F mutation (compare C’ to B’) (arrow). (D) Control homozygous MS1096-gal4 wing. (E) Wing homozygous for MS1096-gal4 and UAS Ras. Extra wing vein material is obvious, particularly where the longitudinal veins meet the wing margin (arrows). (F) Wing homozygous for MS1096-gal4 and UAS Ras. The extra wing vein phenotype (arrows) is enhanced compared to RasWT. (G) Quantification of wing area for wings shown in D-F. “ns” = not significant. ** = p<0.005. Female wings are shown. For male wings, see S3 Fig. Genotypes for wings in this and subsequent figures are indicated in the Methods section.

Y4F substitution enhances oncogenic Ras phenotypes in vivo

Expressing RasG12V in the wing causes wing vein abnormalities (Fig 3B, 3E and 3H; S4B Fig, S4E Fig, S4H Fig) compared to controls (Fig 3A, 3D and 3G; S4A Fig, S4D Fig, S4G Fig). Y4F mutation in oncogenic Ras, RasY4F,G12V, enhanced wing vein abnormalities and further reduced wing size and caused some wings to appear crumpled (Fig 3C, 3F and 3I; S4C Fig, S4F Fig, S4I Fig). Increasing Rabex-5 E3 activity by co-expressing Rabex-5DPYT [13, 31] to a level with no wing phenotype (Fig 3D’; S4D’ Fig) suppressed RasG12V phenotypes (Fig 3E’; S4E’ Fig) but did not suppress RasY4F,G12V phenotypes (Fig 3F’; S4F’ Fig). The enhanced phenotypes of RasY4F,G12V compared to RasG12V and the suppression of RasG12V but not RasY4F,G12V by Rabex-5 E3 activity are consistent with a model that Y4F mutation allows RasG12V to evade Rabex-5-mediated inhibition and emphasize the importance of Ras Y4 for targeting Ras for inhibition.

Fig 3

Non-phosphorylatable oncogenic Ras enhances oncogenic Ras phenotypes.

Increasing temperature increases Gal4/UAS-mediated expression of transgenes. (A) Control wing (c765-gal4/+) at 18°C. (B) Oncogenic Ras, RasG12V, expressed using c765-gal4 at 18°C causes subtle vein abnormalities. (C) Y4F mutation in oncogenic Ras, RasY4F,G12V, shows an increase in wing vein effects. (D) Control wing (c765-gal4/+) at 21°C. (D’) Control wing expressing low level of Rabex-5DPYT using c765-gal4 at 21°C. This level of expression is not sufficient to disrupt wing vein pattern. (E) RasG12V, expressed using c765-gal4 at 21°C, causes extra wing veins and thickened veins. (E’) Rabex-5DPYT expression concurrent to RasG12V, using c765-gal4 at 21°C suppresses the extra wing veins and thickened vein phenotypes. (F) RasY4F,G12V expressed using c765-gal4 at 21°C shows an increase in wing effects including reduction in size compared to RasG12V. (F’) Rabex-5DPYT expression concurrent to RasY4F,G12V using c765-gal4 at 21°C shows a similar phenotype as RasY4F,G12V. (G) Control wing (c765-gal4/+) at 22°C. (H) RasG12V, expressed using c765gal4 at 22°C causes a more severe phenotype than at 21°C. (I) RasY4F,G12V expressed using c765-gal4 at 22°C shows further wing disruption compared to RasG12V. Female wings are shown; for male wings, see S4 Fig.

Negative charge at Y4 suppresses oncogenic Ras phenotypes

If preventing phosphorylation at Y4 allowed Ras to evade inhibition by Rabex-5, then mimicking Y4 phosphorylation would be predicted to increase targeting by Rabex-5, thus inhibiting Ras activity. Notably, RasY4E,G12V exhibited suppressed phenotypes compared to RasG12V in all contexts tested. RasY4E,G12V expression in the early eye with ey-gal4 (Fig 4C, 4E and 4F; S5C Fig, S5E and S5F Fig) resembled a control eye (Fig 4A, 4D and 4F; S5A Fig, S5D Fig, S5F Fig) lacking the RasG12V overgrowth and tissue outgrowths (Fig 4B, 4D and 4E; S5B Fig, S5D and S5E Fig). RasY4E,G12V expression later in eye development with GMR-gal4 (Fig 4I, S5I and S5J Fig) resembled a control eye (Fig 4G, S5G Fig), not a rough RasG12V eye (Fig 4H, S5H–S5J Fig). RasY4E,G12V expressed in hemocytes with He-gal4 (Fig 4L, S5M Fig) resembled controls (Fig 4J, S5K Fig) not RasG12V-induced hemocyte over-proliferation (Fig 4K, S5L Fig). RasY4E,G12V expressed in the wing with c765-gal4 (Fig 4O; S5P Fig) resembled a control wing (Fig 4M; S5N Fig) not the RasG12V wing vein phenotype (Fig 4N; S5O Fig). RasY4E,G12V expressed in the dorsal wing with MS1096-gal4 (Fig 4Q; S5R Fig) suppressed the lethality of RasG12V and caused disrupted wings compared to control wings (Fig 4P; S5Q Fig). These results demonstrate that negative charge at Ras Y4 is a mechanism for inhibiting Ras in vivo; this inhibition is strong enough to block oncogenic Ras visible phenotypes and to prevent oncogenic RasG12V–induced lethality.

Fig 4

Ras Y4 phosphomimic suppresses the phenotypes of oncogenic Ras dependent on the presence of Rabex-5.

(A-F) Y4E phosphomimic mutation suppresses the eye overgrowth and outgrowth phenotypes of RasG12V. Control eye (ey-gal4/+) (A, left eye in D, left eye in F). RasG12V, driven by ey-gal4 (B, right eye in D and E). RasY4E,G12V driven by ey-gal4 (C, left eye in E, right eye in F). Head-to-head photos in D-F highlight the suppression of overgrowth. (G) Control GMR-gal4/+ eye. (H) RasG12V driven by GMR-gal4. (I) RasY4E,G12V driven by GMR-gal4. Y4E phosphomimic mutation suppresses the rough eye and black tissue phenotypes of RasG12V. Female eyes are shown in A-I. For male eyes, see S5A–S5J Fig. (J-L) He-gal4 was used to drive Ras transgene expression in hemocytes. To visualize hemocytes, a UAS GFP transgene was also used. (J) Control, GFP driven by He-gal4. (K). RasG12V and GFP driven by He-gal4. (L). RasY4E,G12V and GFP driven by He-gal4. Larvae in J-L were imaged at the same settings. Tracings of larvae in J and L indicate larval outlines. Excess hemocytes are evident in (K) by the strong GFP signal (green). The excess hemocyte phenotype is suppressed upon Y4E mutation. Scale bars in J-L indicate 1.5 mm. Images of the entire larvae are shown in S5K–S5M Fig. (M) Control wing (c765-gal4/+). (N) RasG12V driven by c765-gal4. (O) RasY4E,G12V driven by c765-gal4. Y4E phosphomimic mutation suppresses the extra wing vein phenotype of RasG12V. (P) Control homozygous MS1096-gal4 wing. (Q) Wing homozygous for MS1096-gal4 and RasY4E,G12V. One copy of oncogenic Ras driven by ms1096gal4 is lethal (therefore wings cannot be shown); Y4E phosphomimic mutation yields obvious wing phenotypes but suppresses the lethality of expressing two copies of RasG12V. (R) Control c765-gal4/+ wing. (S) Wing homozygous for c765-gal4 and RasY4E,G12V transgene show the obvious extra wing vein phenotype associated with oncogenic Ras. (T) Low-level Rabex-5 RNAi driven by c765-gal4 yields no visible phenotype. (U) RasY4E,G12V expression driven by c765-gal4 shows very subtle or no extra wing vein phenotypes. (V) RasY4E,G12V expression elicits obvious extra wing vein phenotypes (arrows) upon concurrent low-level Rabex-5 RNAi driven by c765-gal4. Female wings are shown in M-V; for male wings, see S5N–S5W Fig.

RasY4E expressed with Act5C-gal4 rescued the early lethality of Ras loss-of-function alleles to the same extent as RasWT transgenes (S3I Fig), and RasY4E,G12V expressed in the dorsal wing promoted Ras phenotypes (Fig 4Q; S5R Fig). These data suggest that Y4E mutation does not inherently inactivate Ras, for example by causing misfolding. The ability of negative charge at Y4 to potently block oncogenic Ras activity is consistent with a model that Y4 phosphorylation leads to Ras inhibition by Rabex-5.

Suppression of oncogenic Ras phenotypes by Y4 phosphomimic substitution requires Rabex-5

If Y4E substitution increased Ras targeting by Rabex-5 in vivo as seen in vitro (Fig 1E–1E”), then reducing Rabex-5 gene dosage should elicit oncogenic Ras phenotypes. Increased expression of RasY4E,G12V resulted in obvious oncogenic Ras phenotypes (Fig 4S; S5T Fig) presumably due to overwhelming endogenous Rabex-5. Low-level Rabex-5 RNAi that on its own results in no obvious phenotype (Fig 4T; S5U Fig) and resembles a control wing (Fig 4R; S5S Fig) together with RasY4E,G12V expression resulted in obvious ectopic wing veins (Fig 4V; S5W Fig). The lack of phenotype upon RasY4E,G12V expression (Fig 4O and 4U; S5P Fig, S5V Fig) but extra wing vein phenotype upon concurrent Rabex-5 reduction (Fig 4V; S5W Fig) is consistent with a model that endogenous Rabex-5 strongly restricts RasY4E,G12V activity.

JAK2 and SRC kinases can promote phosphorylation of recombinant RasWT and RasG12V at Y4

No motif or recognition signal responsible for targeting Ras for inhibition by Rabex-5 has previously been identified in Ras. Our findings demonstrate that Rabex-5-mediated inhibition of Drosophila Ras requires conserved Ras residue Tyrosine 4 in vitro and in vivo to regulate Ras. We cannot rule out a requirement for the tyrosine hydroxyl group in protein interactions contributing to this inhibition separate from its role in phosphorylation. However, to more specifically address phosphorylation of Drosophila Ras at Y4, we generated anti-phospho-Y4 (anti-pY4) rabbit polyclonal antibodies (described further in the methods). Anti-pY4 polyclonal antibodies recognized a number of proteins in cell lysates with presumably similar epitopes (S6A Fig), and they also recognized unphosphorylated Ras proteins expressed in and purified from bacteria at a baseline level (Fig 5A and 5B, S6B–S6H Fig). Baseline recognition of unmodified Ras proteins makes it difficult to assess subtle changes in phosphorylation of Y4 from cellular samples. Alanine scanning (S2 Fig) identified a role for Y4 and V7 in ubiquitination of both RasWT and RasG12V; these residues represent a YXXV motif known to be recognized by tyrosine kinases JAK2 [32] and SRC [33-34]. Our alanine scanning also showed a role for E3 in RasG12V but not in RasWT; EY is a motif recognized by EGFR [35]. Recombinant His6-tagged RasWT and RasG12V proteins purified from bacteria were incubated in the presence or absence of purchased recombinant active JAK2, SRC, and EGFR kinases. Reproducibly, anti-p-Y4 antibodies showed a dramatic increase in recognition of RasWT and RasG12V proteins compared to the baseline recognition of non-phosphorylated recombinant protein after incubation with JAK2 and SRC kinases (Fig 5A and 5B; S6B–S6F Fig). Anti-pY4 antibodies did not show a difference in recognition of RasWT protein versus RasWT protein incubated with EGFR kinase (Fig 5A and 5B, S6C Fig, S6E Fig) but showed dramatically increased recognition of RasG12V incubated with specific preparations of EGFR protein (Fig 5A, S6D Fig) but not with all EGFR preparations (Fig 5B, S6F Fig) compared to its recognition of RasG12V protein alone. To confirm that recognition depended on Y4, we repeated these assays with recombinant His6-tagged RasY4F and RasY4F,G12V proteins. Anti-pY4 antibodies did not show a difference in recognition between RasY4F or RasY4F,G12V proteins versus RasY4F or RasY4F,G12V proteins incubated with JAK2, SRC, or EGFR kinases (Fig 5B, S6G and S6H Fig). These findings are consistent with a model that JAK2 and SRC kinases are capable of promoting phosphorylation of RasWT and RasG12V at Y4 in vitro. Our results could also be consistent with a model that EGFR can promote phosphorylation of RasG12V at Y4 in vitro. However, given that this activity varied between preparations of commercial EGFR purified from HEK293 cells, it is possible that this activity relies on a co-purifying factor and is not intrinsic to EGFR. Alternatively, EGFR activity for Ras Y4 may require specific modifications of EGFR or co-factors not consistent between preparations. Anti-pY4 antibodies also recognized JAK2 and SRC kinases to varying degrees depending on specific preparations (S6C–S6H Fig), presumably due to the similar epitopes of their auto-phosphorylation sites with their site in the Ras N-terminus used to generate the anti-pY4 antibodies. Taken together, our in vivo phenotypic data (S3–S5 Figs) and in vitro kinase assay data (Fig 5, S6 Fig) are consistent with a model that the Y4 requirement involves phosphorylation potentially by JAK2, SRC, and/or EGFR tyrosine kinases (summarized schematically in Fig 5J). Serine and threonine phosphorylation have been reported to be required for recognition by the SCF family of cullin ring ligases [36; for review, 37–38]. Reports of tyrosine phosphorylation directing recognition by an E3 are relatively rare. A role for Y4 phosphorylation in inhibition by Rabex-5 could reflect an increased affinity of Rabex-5 for Ras phosphorylated at Y4 as with the SCF and its phosphorylated substrates. Alternatively, a phosphorylated tyrosine could serve another role such as recruitment of an SH2 domain containing adaptor protein that facilitates Ras interaction with Rabex-5.

Fig 5

JAK2, SRC, and EGFR promote Ras phosphorylation at Y4.

(A) RasWT and RasG12V proteins incubated in the presence or absence of JAK2, EGFR, or SRC proteins. Anti-pY4 antibodies recognize a baseline level of recombinant Ras protein species (lane 1). Increased recognition of RasWT protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 2) and SRC (lane 4) but not EGFR (lane 3) compared to unmodified protein (lane 1). Increased recognition of RasG12V protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 2), EGFR (lane 3) and SRC (lane 4) compared to unmodified protein (lane 1). (B) RasWT, RasG12V, RasY4F, and RasY4F,G12V proteins incubated in the presence or absence of different preparations of JAK2, EGFR, or SRC proteins. Increased recognition of RasWT and RasG12V proteins by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5) and SRC (lane 7) but not EGFR (lane 6) compared to unmodified protein (lane 1). No difference in recognition of RasY4F or RasY4F,G12V proteins by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5), EGFR (lane 6) and SRC (lane 7) compared to unmodified protein (lane 1). Anti-pY4 antibodies also recognize JAK2 and SRC (S6B–S6H Fig) but this recognition does not interfere with detection of Ras proteins which run at a different size. (C-C’) Control GMR-gal4/+ eye shown in profile (C) and from overhead (C’). (D-D’) RasG12V expressed using GMR-gal4. Eyes are rough and show some loss of eye pigment. Some eyes have black tissue at the periphery of the eye (arrow) shown in profile (D) and overhead (D’). (E-E-) RasY4F,G12V expressed using GMR-gal4. Some eyes have black tissue at the periphery of the eye (arrow) shown in profile (E) and overhead (E’). (F-F’) hop RNAi driven by GMR-gal4 yields no visible phenotype shown in profile (F) and overhead (F’). (G-G’) hop RNAi concurrent to RasG12V expression using GMR-gal4. Eyes are rough and show a more consistent appearance of black tissue (arrow, quantified in I) shown in profile (G) and overhead (G’). (H-H’) hop RNAi concurrent to RasY4F,G12V expression using GMR-gal4. Eyes are rough but do not show enhancement of the black tissue (arrow, quantified in I) shown in profile (H) or overhead (H’). (I) Graph quantifying the presence of black tissue in control RasG12V and RasY4F,G12V eyes or RasG12V and RasY4F,G12V eyes undergoing concurrent hop RNAi. hop RNAi enhances the appearance of black tissue in RasG12V eyes but not in RasY4F,G12V eyes. In the case shown, hop RNAi suppresses the appearance of black tissue in RasY4F,G12V eyes. Suppression was reproducible but variable; in some trials we saw no statistically significant difference between RasY4F,G12V eyes and RasY4F,G12V eyes undergoing concurrent hop RNAi. Total N is indicated below the graph, and N for each category is indicated in each section of the bar graph.*** indicates p<0.0001, and ** indicates p<0.005 from CHITEST function in Excel for Chi-square statistical analysis comparing the percentage of black tissue between the indicated genotypes. Female eyes are shown in C-H and quantified in I. Increased lethality in males in these experiments resulted in numbers too small for statistical analysis. (J) We propose a model that Ras phosphorylation at Y4 promotes ubiquitination of Ras-GDP and Ras-GTP by Rabex-5. We consistently see greater ubiquitination of RasG12V than of RasWT, and this is also seen for human Ras [12]. This finding, together with additional amino acids affecting ubiquitination of RasG12V than in RasWT (S2B Fig), suggest that there could be one kinase that targets both Ras-GDP and Ras-GTP and a second kinase that also targets Ras-GTP.

Reducing hop levels enhances RasG12V but not RasY4F,G12V

The Y4 kinase would be expected to increase inhibitory ubiquitination of Ras proteins, thus would serve to inhibit Ras biological outputs. JAK2, SRC, and EGFR have all been described to activate Ras signaling in various contexts. If one or more of these kinases also acts as the Y4 kinase to increase Rabex-5-mediated inhibitory Ras ubiquitination, this would serve as a feedback mechanism to ensure precise pathway outputs. Unfortunately, this also means that testing a role for these kinases to inhibit Ras in vivo using genetic interactions is challenging. Expressing EGFR (S7D Fig), RasG12V (Fig 5D and 5D’, S7B Fig), or RasY4F,G12V (Fig 5E and 5E’, S7C Fig) individually in the eye using GMR-gal4 leads to a rough eye, some loss of eye pigment, and in some eyes, the appearance of black tissue around the periphery of the eye compared to a control eye (Fig 5C and 5C’, S7A Fig). Indeed, co-expressing EGFR and RasG12V with GMR-gal4 (S7E Fig) led to a dramatic enhancement of both the EGFR and the RasG12V rough eye phenotypes. Co-expression led to more significant loss of red eye pigment and an increased prevalence of black tissue at the periphery of the eye (S7E Fig). This differs from the phenotype of co-expressing EGFR and RasY4F,G12V which also led to significant loss of eye pigment, but the black tissue phenotype appeared not only at the periphery of the eye but also in other regions of the eye (S7F Fig). The spread of black tissue from the periphery of the eye to other regions of the eye could reflect a greater enhancement of phenotype consistent with a role for EGFR to both restrict RasG12V activity and also to promote signaling through endogenous Ras but an inability to restrict RasY4F,G12V, or could reflect the enhanced phenotype of RasY4F,G12V generally. Thus, these genetic interactions are difficult to interpret.

Low level RNAi of hopscotch (hop, an ortholog of JAK2) in the eye using GMR-gal4 leads to no phenotype on its own (Fig 5F and 5F’) and resembles a control eye (Fig 5C and 5C’). RNAi to hop concurrent to expressing RasG12V (Fig 5G and 5G’, quantified in 5I, raw data in S3 File) consistently enhanced the phenotype of appearance of black tissue in the eye, a quantifiable phenotype we have used previously to reflect severity of phenotype [13] (Fig 5I). Importantly, this enhancement required Y4; we did not see enhancement of this black tissue phenotype upon hop RNAi concurrent to expressing RasY4F,G12V (Fig 5H and 5H’, quantified in 5I). These in vivo interactions taken together with the in vitro kinase assays would be consistent with Hop/JAK2 activity normally acting to promote Y4 phosphorylation to restrict Ras activity in this context.

This report focuses on the importance of a specific tyrosine in directing Rabex-5 mediated ubiquitination of Drosophila Ras (model, Fig 5J). The site of inhibitory ubiquitination in Drosophila Ras and mammalian H-Ras and N-Ras has not been identified. There are a number of solvent-exposed lysines in mammalian Ras proteins reported to be sites of ubiquitination [20, 39–47], many of which are conserved in Drosophila Ras (summarized in S1 Table). K117 mono-ubiquitination has been reported to increase intrinsic nucleotide dissociation which facilitates GDP-GTP exchange, thus activating Ras. K147 mono-ubiquitination has been shown to decrease the rate of GTP hydrolysis mediated by GTPase Activating Proteins [41], which also activates Ras. K170 has been shown to serve as a site of LZTR1-mediated inhibitory ubiquitination [20]. We speculate that one of these other lysines could serve as the biologically relevant site of Rabex-5 mediated inhibitory mono- and di-ubiquitination of Ras proteins. In addition to the potential for inhibitory ubiquitination to promote re-localization of Ras (SS1I Fig and [15]), it is possible that Rabex-5 mediated inhibitory Ras ubiquitination decreases GDP-GTP exchange or increases the rate of GTP hydrolysis in opposition to the activating ubiquitination events at K117 and K147.

Importantly, Y4 mutations in H-Ras (HRasY4H) have been reported in cerebellar glioblastomas [48]. H-RasY4H tumor variants taken together with our work showing gain-of-function phenotypes upon mutation at Y4 suggest that Y4 phosphorylation is important for maintaining appropriate restriction of Ras activity and that mutation at Y4 evades such inhibition to promote tissue transformation.

Materials and methods

Reproducibility

The reported work represents reproducible experiments that reflect a minimum of three well-controlled, independent trials. For phenotypes that are subjective (not quantifiable), independent lab members scored progeny blind to avoid bias.

Tissue culture

S2 cells were cultured using standard methods at 25°C in Schneider’s Drosophila Medium (1X) (Gibco, 21720–024). Cells were transfected using Effectene Transfection Reagent (Qiagen, Cat # 301427) according to manufacturer instructions with plasmids pUAST-HA-Ub, Act-gal4, UAS myc Rabex-5, pIE Flag-His6-GFP Ras WT, pIE Flag-His6-GFP Ras 1-100CKML, pIE Flag-His6-GFP Ras 81–189, pIE Flag-His6-GFP Ras HVR, pIE Flag-His6-GFP Ras 180–189, pIE Flag-His6-GFP Ras 61-80CKML, pIE Flag-His6-GFP Ras 41-60CKML, pIE Flag-His6-GFP Ras 21-40CKML, pIE Flag-His6-GFP Ras 1-20CKML, pIE Flag-His6-GFP Ras 1–10 CKML, pIE Flag-His6-GFP Ras 1–60 CKML, pIE Flag-His6 Ras WT, pIE Flag-His6 Ras M1A, pIE Flag-His6 Ras T2A, pIE Flag-His6 Ras E3A, pIE Flag-His6 Ras Y4A, pIE Flag-His6 Ras Y4E, pIE Flag-His6 Ras Y4F, pIE Flag-His6 Ras Y4F, pIE Flag-His6 Ras E5A, pIE Flag-His6 Ras L6A, pIE Flag-His6 Ras V7A, pIE Flag-His6 Ras V8A, pIE Flag-His6 Ras V9A, pIE Flag-His6 Ras G10A, pIE Flag-His6 Ras M1A,G12V, pIE Flag-His6 Ras E3A,G12V, pIE Flag-His6 Ras Y4A,G12V, Flag-His6 Ras E5A,G12V, Flag-His6 Ras L6A,G12V, Flag-His6 Ras V7A,G12V, Flag-His6 Ras V8A,G12V, Flag-His6 Ras V9A,G12V, Flag-His6 Ras G10A,G12V as indicated and harvested after 48–72 hours. Importantly, amounts of pUAST-HA-Ub and Act-gal4 (to direct ubiquitin over-expression) were kept constant across experiments. Constructs are summarized in Table 1, and protein sequences of Ras constructs are detailed in Table 2.

Table 1

Table of reagents used in the manuscript with corresponding identifiers.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Primary Antibodies
Mouse monoclonal anti-FLAG M2 primary antibodies	Sigma	Catalog #: F1804-5MG
Rabbit polyclonal anti-FLAG primary antibodies	Sigma	Catalog #: F7425-.2MG
Mouse monoclonal anti-HA primary antibodies	Roche	Catalog #: 12CA5
Rat monoclonal anti-Drosophila E Cadherin primary antibodies	Developmental Studies Hybridoma Bank	Catalog #: DCAD2
Mouse monoclonal anti-Pan Ras (AB-3) (Ras 10) primary antibodies	Millipore Sigma	Catalog #: OP40100UG
Mouse monoclonal anti-tubulin primary antibodies, Clone DM1A	Sigma	Catalog #: T9026.-.2ML
Mouse monoclonal anti-phospho-tyrosine primary antibodies, clone 4G10	Millipore Sigma	Catalog #: 05–321
Rabbit polyclonal anti- MTEphosphoYKLVV n(anti-Ras pY4)	Genscript	This study
Secondary antibodies
Goat anti-mouse IgG (H+L) Alexa Fluor 488	Molecular Probes–Invitrogen	Catalog #: A11001
Goat anti-mouse IgG (H+L) Alexa Fluor 680	Molecular Probes–Invitrogen	Catalog #: A21057
Goat anti-rabbit IgG (H+L) Alexa Fluor 680	Molecular Probes–Invitrogen	Catalog #: A21076
Goat anti-mouse IgG (H+L) DyLight 800 Conjugated secondary antibodies	Thermo Scientific	Catalog #: 35521
Goat anti-rabbit IgG (H+L) DyLight 800 conjugated secondary antibodies	Thermo Scientific	Catalog #: 35571
Cell Culture and biochemistry reagents
Qiagen Ni-NTA Agarose	QIAGEN	Catalog # 30230
COMPLETE EDTA free protease inhibitor Cocktail	Roche	Catalog # 11873580001
Phosphosafe Extraction Buffer	Sigma Aldrich	Catalog # 71296–3
Phenylmethylsulfonyl Fluoride	Sigma	Catalog # S-6508
Schneider’s Drosophila Medium (1X)	GIBCO	21720–024
Effectene Transfection Reagent	QIAGEN	Catalog # 301427
Recombinant proteins
JAK2 JH1 active human Jak2 JH1 kinase	Sigma-Aldrich	Catalog # SPR0171-10UG
SRC, active human GST-tagged SRC	Sigma-Aldrich	Catalog #: S1076-10UG
EGFR/ERBB!, Human Egfr/ERBBB1	Sigma-Aldrich	Catalog #: SRP404-10UG
Rosetta-gami B(DE3)PLysS Competent cells	Novagen	Catalog# 71137–4
His6-RasWT (pet-28 RasWT)	This study
His6-RasY4F (pet-28 RasY4F)	This study
His6-RasG12V (pet-28 RasG12V)	This study
His6-RasY4F,G12V (pet-28 RasY4F,G12V)	This study
Drosophila Strains
w1118	The fly community and Bloomington Drosophila Stock Center (BDSC)	BL-3605, BL-5905 and othersRRID:BDSC_3605, RRID:BDSC_5905
Tub-gal4	The NYC fly community	Can be obtained from BDSC, BL-5138,RRID:BDSC_5138
Act5C-gal4	BDSC	BL3954, RRID:BDSC_3954
ms1096-gal4	BDSC	BL-8696RRID:BDSC_8696
c765-gal4	BDSC, NYC fly community	Can be obtained from BDSC, BL-36523RRID:BDSC_36523
Ey-gal4	NYC fly community
GMR-gal4	BDSC	BL-8605, RRID:BDSC_8605
He-gal4; UAS GFP.nls	BDSC	BL-8700, RRID:BDSC_8700
UAS hopIR (P{TRiP.JF01267})	BDSC	BL-31699; RRID:BDSC_31699
UAS EGFR	BDSC	BL-9535; RRID:BDSC_9535
UAS Rabex-5IR (P{GD14133}v46329 CG9139GD14133)	VDRC	VDRCID dna14133
UAS Rabex-5DPYT	Yan et al. 2010 [13]
UAS Flag-His6-RasWT	This study
UAS Flag-His6-RasY4E	This study
UAS Flag-His6-RasY4F	This study
UAS Flag-His6-RasG12V	This study
UAS Flag-His6-RasY4E,G12V	This study
UAS Flag-His6-RasY4F,G12V	This study
Experimental Models: Cell Lines
Drosophila S2 cells	A gift from the NYC fly community
Recombinant DNA
pUAST-HA-Ub	Yan et al. 2010
Act-gal4	A gift from the Mlodzik lab, also used in Yan et al. 2010
UAS myc Rabex-5	Yan et al. 2010
pIE1-4 Flag-His6-GFP Ras WT	This study
pIE1-4 Flag-His6-GFP Ras 1-100CKML	This study
pIE1-4 Flag-His6-GFP Ras 81–189	This study
pIE1-4 Flag-His6-GFP Ras HVR	This study
pIE1-4 Flag-His6-GFP Ras 180–189	This study
pIE1-4 Flag-His6-GFP Ras 61-80CKML	This study
pIE1-4 Flag-His6-GFP Ras 41-60CKML	This study
pIE1-4 Flag-His6-GFP Ras 21-40CKML	This study
pIE1-4 Flag-His6-GFP Ras 1-60CKML	This study
pIE1-4 Flag-His6-GFP Ras 1-20CKML	This study
pIE1-4 Flag-His6-GFP Ras 1–10 CKML	This study
pIE1-4 MYC GFP 1–20 CKML	This study
pIE1-4 MYC GFP 61–80 CKML	This study
pIE1-4 Flag-His6-GFP Ras2	This study
pIE1-4 Flag-His6 Ras WT	Yan et al. 2010
pIE1-4 Flag-His6 Ras M1A	This study
pIE1-4 Flag-His6 Ras T2A	This study
pIE1-4 Flag-His6 Ras E3A	This study
pIE1-4 Flag-His6 Ras Y4A	This study
pIE1-4 Flag-His6 Ras Y4E	This study
pIE1-4 Flag-His6 Ras Y4F	This study
pIE1-4 Flag-His6 Ras E5A	This study
pIE1-4 Flag-His6 Ras L6A	This study
pIE1-4 Flag-His6 Ras V7A	This study
pIE1-4 Flag-His6 Ras V8A	This study
pIE1-4 Flag-His6 Ras V9A	This study
pIE1-4 Flag-His6 Ras G10A	This study
pIE1-4 Flag-His6 Ras G12V	This study
pIE1-4 Flag-His6 Ras M1A,G12V	This study
pIE1-4 Flag-His6 Ras T2A,G12V	This study
pIE1-4 Flag-His6 Ras E3A,G12V	This study
pIE1-4 Flag-His6 Ras Y4A,G12V	This study
pIE1-4 Flag-His6 Ras K5A,G12V	This study
pIE1-4 Flag-His6 Ras L6A,G12V	This study
pIE1-4 Flag-His Ras V7A,G12V	This study
pIE1-4 Flag-His6 Ras V8A,G12V	This study
pIE1-4 Flag-His6 Ras V9A,G12V	This study
pIE1-4 Flag-His6 Ras G10A,G12V	This study
pet28-RasWT	This study
pet28-RasY4F	This study
pet28-RasG12V	This study
pet28-RasY4F,G12V	This study
Software
Image J		https://imagej.nih.gov/ij/
Adobe Photoshop		https://www.adobe.com/products/photoshop.html
GraphPad Prism		https://www.graphpad.com/scientific-software/prism/
Microsoft Excel		https://www.microsoft.com/Microsoft/Excel/

Table 2

Table of protein sequences for Ras constructs used in vitro and in vivo.

Construct (as listed in Table 1)	Protein sequence (mutations bold, underlined; Tags and CAAX box, bolded): FLAG = DYKDDDDK, yellow His6 = HHHHHH GFP = MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK MYC = EQKLISEEEDL Ras CAAX Box = CKML
pet28-RasWT	MGSSHHHHHHSSGENLYFQGRPMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pet28-RasY4F	MGSSHHHHHHSSGENLYFQGRPMTEFKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pet28-RasG12V	MGSSHHHHHHSSGENLYFQGRPMTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pet28-RasY4F,G12V	MGSSHHHHHHSSGENLYFQGRPMTEFKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6-GFP Ras WT	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6-GFP Ras 1-100CKML	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQICKML
pIE1-4 Flag-His6-GFP Ras 81–189	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6-GFP Ras HVR	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6-GFP Ras 180–189	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEPNRRFKCKML
pIE1-4 Flag-His6-GFP Ras 61-80CKML	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEQEEYSAMRDQYMRTGEGFLLCKML
pIE1-4 Flag-His6-GFP Ras 41-60CKML	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALERKQVVIDGETCLLDILDTAGCKML
pIE1-4 Flag-His6-GFP Ras 21-40CKML	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEIQLIQNHFVDEYDPTIEDSYCKML
pIE1-4 Flag-His6-GFP Ras 1-60CKML	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGCKML
pIE1-4 Flag-His6-GFP Ras 1-20CKML	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEMTEYKLVVVGAGGVGKSALTCKML
pIE1-4 Flag-His6-GFP Ras 1–10 CKML	MDYKDDDDKRGSHHHHHHAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKALEMTEYKLVVVGCKML
pIE1-4 MYC GFP 1–20 CKML	MEQKLISEEDLAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGLEMTEYKLVVVGAGGVGKSALTCKML
pIE1-4 MYC GFP 61–80 CKML	MEQKLISEEDLAMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGLEQEEYSAMRDQYMRTGEGFLLCKML
pIE1-4 Flag-His6 Ras WT	MDYKDDDDKRGSHHHHHHALEMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras M1A	MDYKDDDDKRGSHHHHHHALEATEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras T2A	MDYKDDDDKRGSHHHHHHALEMAEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras E3A	MDYKDDDDKRGSHHHHHHALEMTAYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras Y4A	MDYKDDDDKRGSHHHHHHALEMTEAKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras Y4E	MDYKDDDDKRGSHHHHHHALEMTEEKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras Y4F	MDYKDDDDKRGSHHHHHHALEMTEFKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras E5A	MDYKDDDDKRGSHHHHHHALEMTEYALVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras L6A	MDYKDDDDKRGSHHHHHHALEMTEYKAVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras V7A	MDYKDDDDKRGSHHHHHHALEMTEYKLAVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras V8A	MDYKDDDDKRGSHHHHHHALEMTEYKLVAVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras V9A	MDYKDDDDKRGSHHHHHHALEMTEYKLVVAGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras G10A	MDYKDDDDKRGSHHHHHHALEMTEYKLVVVAAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras G12V	MDYKDDDDKRGSHHHHHHALEMTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras M1A,G12V	MDYKDDDDKRGSHHHHHHALEATEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras T2A,G12V	MDYKDDDDKRGSHHHHHHALEMAEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras E3A,G12V	MDYKDDDDKRGSHHHHHHALEMTAYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras Y4A,G12V	MDYKDDDDKRGSHHHHHHALEMTEAKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras K5A,G12V	MDYKDDDDKRGSHHHHHHALEMTEYALVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras L6A,G12V	MDYKDDDDKRGSHHHHHHALEMTEYKAVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His Ras V7A,G12V	MDYKDDDDKRGSHHHHHHALEMTEYKLAVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras V8A,G12V	MDYKDDDDKRGSHHHHHHALEMTEYKLVAVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras V9A,G12V	MDYKDDDDKRGSHHHHHHALEMTEYKLVVAGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
pIE1-4 Flag-His6 Ras G10A,G12V	MDYKDDDDKRGSHHHHHHALEMTEYKLVVVAAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
UAS Flag-His6-RasWT	MDYKDDDDKRGSHHHHHHALEMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
UAS Flag-His6-RasY4E	MDYKDDDDKRGSHHHHHHALEMTEEKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
UAS Flag-His6-RasY4F	MDYKDDDDKRGSHHHHHHALEMTEFKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
UAS Flag-His6-RasG12V	MDYKDDDDKRGSHHHHHHALEMTEYKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
UAS Flag-His6-RasY4E,G12V	MDYKDDDDKRGSHHHHHHALEMTEEKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML
UAS Flag-His6-RasY4F,G12V	MDYKDDDDKRGSHHHHHHALEMTEFKLVVVGAVGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLLVFAVNSAKSFEDIGTYREQIKRVKDAEEVPMVLVGNKCDLASWNVNNEQAREVAKQYGIPYIETSAKTRMGVDDAFYTLVREIRKDKDNKGRRGRKMNKPNRRFKCKML

Schneider S2 cell extract preparation

Schneider S2 cell extracts were prepared immediately from harvested cells or from frozen cell pellets. Lysates were prepared in lysis buffer, 1X NP40 buffer (50 mM HEPES pH 7.4, 1% NP40, 1mM EDTA, 150 mM NaCl), 8M Urea, 5 mM beta-mercaptoethanol, 10 mM imidazole supplemented with Roche protease inhibitor cocktail and 1mM each of PMSF and Sodium Orthovanadate) and used in pull down assays described above or analyzed by Western.

Nickel pull down of tagged Ras from Schneider S2 cells (for Western analysis)

Ni-NTA agarose beads (Qiagen) were equilibrated as per manufacturer’s instructions and then washed twice with binding buffer, pH 8.0 (1X PBS, 8 M Urea, 0.5% NP40, 10 mM Imidazole, 360 mM NaCl) followed by incubation with binding buffer, pH 8.0 supplemented with 1g/ml BSA. Lysates were then incubated with beads on a nutator for 2h at 4°C. Beads were then transferred to mini-columns on a vacuum assembly and washed 3X with wash buffer pH 6.3 (1X PBS, 8 M Urea, 0.5% NP40, 30 mM Imidazole, 360 mM NaCl). Purified His-tagged proteins were eluted from the beads by washing with elution buffer pH 6.3 (1X PBS, 8 M Urea, 0.5% NP40, 500 mM Imidazole, 360 mM NaCl). Eluate was boiled in 1X loading buffer for 10 minutes before storage at -20°C.

Larval extract preparation

Extracts were prepared from larvae of the specified genotypes in Phosphosafe Extraction Reagent (Novagen) supplemented with protease inhibitors (Complete Phosphatase Inhibitor Cocktail, 1mM PMSF and 1mM Sodium Orthovanadate). Lysates of individual larvae were loaded per lane to demonstrate transgene protein levels.

Development of anti-phospho-Y4 antibodies

A project was initiated with Genscript to produce MTE{pY}KLVVVGC peptides to immunize rabbits. Genscript immunized rabbits and purified phospho-specific polyclonal antisera which recognized MTE{pY}KLVVVGC peptides and delivered purified antibodies to us following purification. Antibodies were tested against cell lysates, larval lysates, and purified recombinant proteins.

Western Blot analysis

Western blots used Immobilon-FL Transfer Membrane (Millipore, Cat # IPFL00010) and were visualized using the Li-Cor Odyssey System. Primary antibodies were anti-pY4 (rabbit polyclonal, this study; 1:2000); anti-FLAG (rabbit, Sigma Catalog # F7425-.2MG, 1:1000), anti-Pan Ras (mouse, Millipore Sigma Catalog # OP40100UG, 1:1000), anti-FLAG M2 (mouse, Sigma Catalog # F1804-5MG, 1:1000), anti-HA (mouse, Roche Catalog # 12CA5, 1:1000), anti-alpha tubulin (mouse, Sigma T9026.-.2ML, 1:8000), anti-phospho-tyrosine 4G10 (mouse, EMD Millipore Catalog # 05–321, 1:1000); secondary antibodies were Alexa Fluor goat anti-mouse 488 (Invitrogen, Catalog # A11001, 1:10,000), Alexa Fluor goat anti-rabbit 680 (Invitrogen, Catalog # A21076, 1:10,000), Alexa Fluor goat anti-mouse 680 (Invitrogen, Catalog # A21057, 1:10,000), DyLight goat anti-mouse 800 (Thermo Scientific, Catalog # 35521, 1:10,000), and DyLight goat anti-rabbit 800 (Thermo Scientific, Catalog # 35571, 1:10,000).

Recombinant proteins

RasWT and Ras mutants (RasY4F, RasG12V, and RasY4F,G12V) were cloned into pet-28 vectors for bacterial expression and purification of His6-tagged proteins. Proteins were expressed from these plasmids in Rosetta-gami B(DE3)PLysS strains (Novagen, 711374) following induction by IPTG. Proteins were purified on nickel beads. Protein sequences of Ras mutants are detailed in Table 2.

Kinase assays

Active JAK2 (SRP0171), EGFR (SRP6404), and SRC (S1076) recombinant proteins were purchased from Sigma. Recombinant RasWT or Ras mutants on beads were incubated in the presence or absence of 100 micrograms (S6B Fig) or 20 micrograms (Fig 5A and 5B, S6C–S6H Fig) recombinant kinase proteins in kinase assay buffer (25mM TrisHCl pH7.5, 10mM MgCl2) for thirty minutes, quenched with sample buffer, run on a gel, and analyzed by Western.

Western quantification and adjustment

Raw tiff files from the Li-Cor Odyssey were split from full color into specific channels corresponding to single antibodies that were then converted to grayscale using Adobe Photoshop. Images were cropped to fit figure panels; adjustments to brightness and contrast were applied uniformly to the entire images not to portions of an image. Quantification of percent ubiquitination utilized gels in which signals were not over-saturated. To quantify percent ubiquitination, gel lanes were analyzed using Image J; we summed the signal of unconjugated and ubiquitinated Ras bands in a single lane to define total Ras signal in that lane and then calculated the proportion of each band per total to indicate percent conjugated to ubiquitin out of total Ras. In this manner, we could make comparisons of percent conjugation to ubiquitin in one lane to the percent conjugation to ubiquitin in another lane from different lanes on the same gel within the same experiment.

Drosophila experiments

Ras constructs were cloned into pUAST-attB with the FLAG and His6 sequences of MDYKDDDDKRGSHHHHHHALE preceding the Ras coding sequence. UAS Flag-His6-RasWT, UAS Flag-His6-RasY4E, UAS Flag-His6-RasY4F, UAS Flag-His6-RasG12V, UAS Flag-His6-RasY4E,G12V, and UAS Flag-His6-RasY4F,G12V plasmids were sent to BestGene for injection and generation of transgenic lines at the attp40 locus. Lines were balanced over CyO or SM6-TM6B balancers and then maintained as true-breeding homozygous stocks. Genomic DNA was sequenced to confirm each insert. Gal4 drivers were obtained from the Bloomington Drosophila Stock center or other labs in the Drosophila community. UAS hop (P{TRiP.JF01267}), UAS EGFR were from the Bloomington Stock center. UAS Rabex-5IR (P{GD14133}v46329 CG9139GD14133) was obtained from the VDRC, VDRCID dna14133 and was characterized in our previous study [13]. Crosses were performed at the indicated temperatures on standard Drosophila medium. Raw wing images were converted to grayscale using Adobe Photoshop. Brightness and contrast of eye and wing images were adjusted using Adobe Photoshop to maximize clarity; adjustments were applied to the entire images. Genotypes are summarized below, and identifiers are annotated in Table 1. Protein sequences for Ras transgenes are detailed in Table 2.

Genotypes of fly images (Figures and Supplemental Figures):

w; Tub-gal4/+ (Fig 2A and 2A’; S3C Fig, S3C’ Fig)

w; UAS Ras/+; Tub-gal4/+ (Fig 2B and 2B’; S3D Fig, S3D’ Fig)

w; UAS Ras/+; Tub-gal4/+ (Fig 2C and 2C’; S3E Fig, S3E’ Fig)

MS1096-gal4 (Fig 2D; S2F Fig; Fig 4P, S5Q Fig)

MS1096-gal4; UAS Ras (Fig 2E; S3G Fig)

MS1096-gal4; UAS Ras (Fig 2F; S3H Fig)

w; c765-gal4/+ (Fig 3A, 3D and 3G, S3A Fig, S3D Fig, S3G Fig; Fig 4M and 4R, S5N Fig, S5S Fig)

w; UAS Ras/+; c765-gal4/+ (Fig 3B, 3E and 3H, S4B Fig, S4E Fig, S4H Fig; Fig 4N, S5O Fig)

w; UAS Rabex-5/+; c765gal4/+ (Fig 3D’, S4D’ Fig)

w; UAS Ras/Rabex-5; c765gal4/+ (Fig 3E’, S4E’ Fig)

w; UAS Ras/+; c765gal4/+ (Fig 3C, 3F and 3I, S4C Fig, S4F Fig, S4I Fig)

w; UAS Ras/Rabex-5; c765-gal4/+ (Fig 3F’, S4F’ Fig)

w; ey-gal4/+ (Fig 4A, left eye in 4D and 4F, S5A Fig, left eye in S5D Fig, S5F Fig)

w; ey-gal4/UAS Ras (Fig 4B, right eye in 4D and 4E, S5B Fig, right eye in S5D Fig, S5E Fig)

w; ey-gal4/UAS Ras (Fig 4C, left in in 4E, right eye in 4F, S5C Fig, left eye in S5E Fig, right eye in S5F Fig)

w; GMR-gal4/+ (Fig 4G, Fig 5C and 5C’, S1H–S1H” Fig, S5G Fig, S7A Fig)

w; GMR-gal4/UAS Ras (Fig 4H, Fig 5D and 5D’, S5H Fig, right eye in S5J Fig, S7B Fig)

w; GMRgal4/ UAS Ras (Fig 4I, S5I Fig, left eye in S5J Fig)

w; He-gal4, UAS GFP.nls/+ (Fig 4J, S5K Fig)

w; UAS Ras/+UAS GFP.nls/+ (Fig 4K, S5L Fig)

w; UAS Ras/+; UAS GFP.nls/+ (Fig 4L, S5M Fig)

MS1096-gal4; UAS Ras (Fig 4Q, S5R Fig)

w; UAS Ras/+; c765-gal4/+ (Fig 4O and 4U, S5P Fig, S5V Fig)

w; UAS Ras; c765-gal4 (Fig 4S, S5T Fig)

w; UAS Rabex-5/+; c765-gal4/+ (Fig 4T, S5U Fig)

w; UAS Rabex-5/ UAS Ras; c765gal4/+ (Fig 4V, S5W Fig)

w; GMR-gal4/+; UAS hop/+ (Fig 5F and 5F’)

w; GMR-gal4/UAS Ras; UAS hop/+ (Fig 5G and 5G’)

w; GMR-gal4/UAS Ras; UAS hop/+ (Fig 5H and 5H’)

w; GMR-gal4/+; UAS Rabex-5/+ (S1I–S1I” Fig)

w; GMR-gal4/UAS Ras (Fig 5E and 5E’, S7C Fig)

w; GMR-gal4/UAS Ras; UAS EGFR/+ (S7C Fig)

w; GMR-gal4/UAS Ras; UAS EGFR (S7C Fig)

Genotypes of flies in Western blots:

w; UAS Ras/+; Act5C-gal4/+ (S3B Fig lane 1)

w; UAS Ras/+; Act5C-gal4/+ (S3B Fig lane 2)

w; UAS Ras/+; Act5C-gal4/+ (S3B Fig lane 3)

Statistical analysis

Wings were measured using Image J software. Wing size comparisons were analyzed using GraphPad Prism software. Unpaired T-tests were used to compare two groups (e.g. controls versus RasY4F over-expressing wings), and one way ANOVA analysis was used for experiments considering three groups (e.g. control versus RasWT versus RasY4F). Chi square analysis (using the CHITEST function in Microsoft Excel was used to compare percentages in Fig 1E–1E” for percent of Ras constructs conjugated to ubiquitin and in Fig 5I to compare the percentage of eyes with black tissue. The expected values for each comparison were calculated based on control values applied to the N for other samples.

An N-terminal Tyrosine-based signal directs Ras for mono- and di-ubiquitination.

(A) Drosophila Ras and H-, N-, and K-Ras share significant homology in their N-termini but differ substantially in a C-terminal region called the HyperVariable Region (HVR). The HVR differs between H-, N, and K-Ras and directs the specific localization of each isoform [19-21]. Schematic showing a series of deletions tested for ubiquitination of Drosophila Ras in S2 cells indicating which deletion constructs supported Ras mono- and di-ubiquitination in S2 cells. Deletion constructs were tagged with FLAG, His6, and GFP at the N-terminus (not depicted). To ensure that C-terminal deletions maintained appropriate localization, all constructs deleting the C-terminus maintained the C-terminal CAAX localization signal, CKML (shown in red). * indicates that this construct in some experiments showed poly-ubiquitination (sample gel shown in D). (B-D) Sample gels corresponding to many of the constructs in the schematic in (A). Specific constructs are indicated above the gel according to the abbreviations listed in (A) for constructs A-K. (B-B’) N- and C-terminal deletions show ubiquitination pattern of full length Ras for only the N-terminal construct. (B) Un-adjusted gels. (B’) Gels from (B) were adjusted to highlight the mono- and di-ubiquitination pattern (or lack thereof); brightness and contrast adjustments were applied to the entire images. (C-C’) 20 amino acid constructs in the N-terminal 80 amino acids for low levels of expression (C) and in over-loaded conditions (C’). Only the N-terminal 20 amino acids (construct J) consistently shows ubiquitin conjugates. Mono- and di-ubiquitin conjugates are never seen for 21–40 and 41–60, and never predominate for 61–80 even for high levels of expression (C’). (D) More than once, we saw poly-ubiquitin conjugates for the tagged 61–80 region (construct G), shown here in comparison to 1–60 which gives the standard Ras pattern of predominantly mono- and di-ubiquitin conjugates. This was seen multiple times, but was not consistent. This may mean that a degradation signal for Nedd4, βTRCP, and LZTR1 could lie in this region, or this could be an artefact of exposing a cryptic degron. We have not resolved this as this was outside the scope of this work. We include this here for transparency. (E) DNA encoding competitive peptides of 1-20CKML or 61-80CKML tagged with MYC and GFP were transfected at the same levels (1X) as RasWT or in five-fold excess (5X). Consistently, over-expression of the 1-20CKML peptide but not the 61-80CKML peptide inhibited formation of RasWT ubiquitin conjugates. (F) Another example highlights the consistency of ubiquitination of 1–10 and 1–20 but not 61–80. (G) Larger gel of cropped images from Fig 1A. The bands recognized by both anti-FLAG and anti-HA antibodies represent ubiquitinated species of Ras (marked by an asterisk, *). Other bands in the anti-HA gel reflect non-Ras, co-purifying ubiquitinated proteins. (H-I”) Pupal eyes dissected 48 hours after puparium formation were stained with antibodies to E-cadherin (Ecad, blue in H-H’ and I-I’; DSHB, catalog # DCAD2, rat monoclonal primary antibodies; goat anti-Rat Alexa Fluor 647 Invitrogen, Catalog # A21247 secondary antibodies) and anti-Pan Ras antibodies to recognize endogenous Ras (red in H, H”, I, I”; Millipore Sigma, catalog # OP40100UG, mouse monoclonal primary antibodies; goat anti-mouse Alexa Fluor 555 Invitrogen, Catalog #A21422 secondary antibodies). (H-H”) Staining of control GMR-gal4/+ pupal eyes shows the pattern of Ecad (blue) and endogenous Ras (red) in the pupal eye. Merge shown in H. (I-I”) Staining of Rabex-5DPYT expressing pupal eyes shows redistribution of Ras (red) to an internal compartment. Merge in I. Boxes in H-I” represent 50 micron areas.

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Ras Tyrosine 4 is important for Ras ubiquitination.

(A-A’) Sample gels showing ubiquitin conjugates of alanine substitution mutants. (A) Gel showing M1A, T2A, E3A, Y4A, K5A, L6A, and V7A mutants compared to control transfected cells (lane 1) and control RasWT (lane 2). Reproducibly, we see decreased ubiquitination for RasY4A and RasV7A mutants (red boxes). We see no decrease or no reproducible decrease for other alanine substitution mutants. (A’) Gel showing E3A, Y4A, K5A, L6A, V7A, V8A, and V9A in the RasG12V context compared to control RasWT (lane 2) and control RasG12V (lane 3) or control-transfected cells (lane 1). Typically, RasG12V shows greater ubiquitin conjugation than RasWT (lane 3 compared to lane 2). This gel is at saturation for RasWT, therefore this may be an underestimate of the increased ubiquitination of RasG12V. (B) Schematic summarizing the results of alanine scanning of the first 10 amino acids of Ras (in the context of full length RasWT or RasG12V) highlighting substitution mutants for which we saw decreased ubiquitination reproducibly. Alanine substitution of Y4 and V7 in otherwise wild-type RasWT (which is primarily in the GDP-loaded conformation) reproducibly decreased ubiquitination. Alanine substitution at E3, Y4, K5, and V7 in RasG12V shows decreased ubiquitination compared to RasG12V (which is in the GTP-loaded conformation). We could not address the role of G10; RasG10A and RasG10A,G12V mutants mislocalized within the cell (H for RasG10A below). (C) Alignment showing complete conservation of the N-terminal 10 amino acids of Drosophila Ras and human H-Ras, N-Ras, and K-Ras. Alignment also shows conservation of the tyrosine and valine in Drosophila Ras2. (D) Gel showing Rabex-5 mediated increase in ubiquitin conjugates for Drosophila RasWT and also for Drosophila Ras2. (E-M) FLAG-His6 tagged Ras alanine mutants that decreased ubiquitination showed localization to the membrane and association in intracellular puncta as did FLAG-His6 RasWT and FLAG-His6 RasG12V controls suggesting that the decreased ubiquitination did not result from inappropriate localization. (E-E”) Typically, Ras is seen at the membrane and at intracellular puncta. Staining a population of cells transfected with RasWT (green, reflecting FLAG staining) reveals individual cells with more membrane-associated Ras (E), Ras at the membrane and in intracellular puncta (E’) or Ras enriched in intracellular puncta (E”). Populations of cells (>100 per sample) were scored blind by multiple lab members to ensure appropriate localization was confirmed. Ras localization is shown for (E-E”) RasWT, (F) RasY4A, (G) RasV7A, (H) RasG10A, (I) RasG12V, (J) RasE3A,G12V, (K) RasY4A,G12V, (L) RasK5A,G12V, and (M) RasV7A,G12V. RasG10A mutants mislocalized (H) so their ubiquitination could not be appropriately interpreted. RasY4A, RasV7A,. RasE3A,G12V, RasY4A,G12V, RasK5A,G12V, and RasV7A,G12V mutants show localization to the membrane and intracellular puncta where RasWT and RasG12V localize, so the decreased ubiquitination did not result from a failure to localize to these compartments in the cell. Boxes in E-M represent 20 μm square regions. (N) S2 cells were transfected with empty vector or FLAG-His6 tagged Ras. Nickel pulldowns from cell lysates showed no recognition by anti-phospho-tyrosine (anti-ptyr) antibodies or anti-FLAG antibodies for vector-control lysates (lane 1) but showed a signal for RasWT lysates confirming that Drosophila Ras is tyrosine phosphorylated. (O-P) FLAG-His6 Ras purified from S2 cells using nickel beads is recognized by anti-ptyr antibodies (lane 1). Phenylalanine substitution mutant RasY4F reproducibly shows decreased recognition (lane 2). Total Ras is shown by anti-FLAG antibodies. Quantification is done by normalizing for the amount of Ras pulled down. Quantification of a sample gel a the upper end of the range (O) shows decreased recognition of RasY4F compared to RasWT by approximately 27% and a sample gel at the lower end of the rage (P) shows a decrease by 17%. Gels at either end of the range are shown for transparency. Typically, we see Ras isolated from cells run as a doublet (obvious in O) although this can be less obvious depending on the separation on the gel.

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(A) Summary table of various gal4 drivers used to express RasWT. In many cases, RasWT expression does not cause an obvious, visible phenotype. (B) Expressing FLAG-His6 tagged RasWT, RasY4E, or RasY4F transgenes using Act5C-gal4 results in similar expression levels. Western blot shows anti-FLAG (the tag on Ras transgenes) and anti-tubulin loading control. (C) Control wing (Tub-gal4/+). (D) RasWT expression driven by Tub-gal4. Ectopic longitudinal vein material is seen anterior to the L2 longitudinal vein (arrow, enlarged in D’) and on the posterior crossvein (arrow). (E) RasY4F expression driven by Tub-gal4. Ectopic longitudinal vein material is seen anterior and posterior to the L2 longitudinal vein (arrow, enlarged in E’). The ectopic wing vein phenotype (arrows) is enhanced upon Y4F mutation (compare E’ to D’). (F) Control homozygous MS1096-gal4 wing. (G) Wing homozygous for MS1096-gal4 and UAS Ras. Extra wing vein material is obvious, particularly where the longitudinal veins meet the wing margin (arrows). (H) Wing homozygous for MS1096-gal4 and UAS Ras. The extra wing vein phenotype (arrows) is enhanced compared to RasWT. Male wings are shown in C-H. (I) Summary table of transgene rescue experiments. Expressing FLAG-His6 tagged RasWT, RasY4E, or RasY4F transgenes using Act5C-gal4 rescues the early lethality of Rase1b/Rase1b; flies survive to the pupal stage.

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(A) Control wing (c765-gal4/+) at 18°C. (B) RasG12V expressed using c765-gal4 at 18°C causes subtle wing effects. (C) RasY4F,G12V shows an increase in wing vein effects. (D) Control wing (c765-gal4/+) at 21°C. (D’) Control wing expressing low level of Rabex-5DPYT using c765-gal4 at 21°C causes no wing vein disruption. (E) RasG12V expressed using c765-gal4 at 21°C causes extra wing veins and thickened veins. (E’) Rabex-5DPYT expression concurrent to RasG12V using c765-gal4 at 21°C suppresses the extra wing veins and thickened vein phenotypes. (F) RasY4F,G12V expressed using c765-gal4 at 21°C shows an increase in wing effects including reduction in size compared to RasG12V. (F’) Rabex-5DPYT expression concurrent to RasY4F,G12V using c765-gal4 at 21°C shows a similar phenotype as RasY4F,G12V. (G) Control wing (c765-gal4/+) at 22°C. (H) RasG12V expressed using c765-gal4 at 22°C causes a more severe phenotype than at 21°C. (I) RasY4F,G12V expressed using c765-gal4 at 22°C shows further wing disruption compared to RasG12V. Male wings are shown.

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(A-F) Y4E phosphomimic mutation suppresses the eye overgrowth and outgrowth phenotypes of RasG12V. Control eye (ey-gal4/+) (A, left eye in D, left eye in F). Oncogenic Ras, RasG12V, driven by ey-gal4 (B, right eye in D and E). RasY4E,G12V driven by ey-gal4 (C, left eye in E, right eye in F). Head-to-head photos in D-F highlight the suppression of overgrowth. (G) Control GMR-gal4/+ eye. (H) Oncogenic Ras, RasG12V, driven by GMR-gal4. (I) RasY4E,G12V driven by GMR-gal4. Y4E phosphomimic mutation suppresses phenotypes of RasG12V. (J) Overhead shot showing a RasY4E,G12V driven by GMR-gal4 (left head) and a RasG12V driven by GMR-gal4 (right head). Arrows indicate small spots of black tissue in the RasG12V eyes that are absent in the morphologically normal RasY4E,G12V eyes. Male eyes are shown in A-J. (K-M) He-gal4 was used to drive Ras transgene expression in hemocytes. To visualize hemocytes, a UAS GFP transgene was also used. The entire larvae from images in Fig 4J–4L are shown. With this driver, there is strong background fluorescence in the salivary glands in the anterior region of the larva (cropped out of the panel in the main figure). (K) Control, GFP driven by He-gal4. (L) RasG12V and GFP driven by He-gal4. (N) RasY4E,G12V and GFP driven by He-gal4. Larvae in K-M were imaged at the same settings. Tracings of larvae in K and M indicate larval outlines. Excess hemocytes are evident in (L) by the strong GFP signal (green). The excess hemocyte phenotype is suppressed upon Y4E mutation. Scale bars in K-M indicate 1.5 mm. (N) Control wing (c765-gal4/+). (O) Oncogenic Ras, RasG12V, driven by c765-gal4. (P) RasY4E,G12V driven by c765gal4. Y4E phosphomimic mutation suppresses the extra wing vein phenotype of RasG12V. (Q) Control homozygous MS1096-gal4 wing. (R) Wing homozygous for MS1096-gal4 and RasY4E,G12V. Oncogenic Ras driven by MS1096-gal4 is lethal; Y4E phosphomimic mutation yields obvious wing phenotypes but suppresses the lethality of one copy or two copies of RasG12V. (S) Control c765-gal4/+ wing. (T) Wing homozygous for c765-gal4 and RasY4E,G12V transgene shows the obvious extra wing vein phenotype associated with oncogenic Ras. (U) Low-level Rabex-5 RNAi driven by c765-gal4 yields no visible phenotype. (V) RasY4E,G12V expression driven by c765-gal4 shows very subtle or no extra wing vein phenotypes. (W) Low-level Rabex-5 RNAi elicits obvious extra wing vein phenotypes (arrows) of RasY4E,G12V expression driven by c765-gal4. Male wings are shown in N-W.

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(A) Gel of Schneider S2 cell extracts probed with anti-pY4 antibodies. The peptide polyclonal anti-Y4 antibodies recognize many bands in non-transfected control cells (“empty,” lane 2), in RasWT-transfected cells (lane 3), and in RasG12V-tranfected cells (lane 4). Molecular weight markers (lane 1) indicate 15, 25, 35, and 55 kDa protein sizes; given the recognition of so many bands by the anti-pY4 antibodies, it is impossible to distinguish over-expressed Ras proteins from other cross-reacting proteins from cell extracts. (B) Recombinant RasWT protein purified from bacteria was incubated in the presence (lane 2) or absence (lane 1) of 100 micrograms of recombinant JAK2. Despite low-level recognition of unmodified RasWT protein in lane 1, there is an obvious dramatic increase in recognition of RasWT protein when incubated with JAK2 in lane 2 (anti-pY4 antibodies, upper gel; anti-Pan Ras antibodies, lower gel). Because the recognition was so dramatic, we were concerned that any cross-reaction with JAK2 itself could not be distinguished from Ras. Therefore, we titrated the amount of JAK2 in kinases assays and could reliably see a response using 20 micrograms of kinase used in subsequent assays. Using a lower level of kinase allowed us to distinguish modified Ras from cross-reacting JAK2 and SRC shown in subsequent panels. (C-D) Full gel of gel slices shown in Fig 5A. (C) RasWT protein incubated in the presence or absence of JAK2, EGFR, or SRC proteins. Increased recognition of RasWT protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 2) and SRC (lane 4) but not EGFR (lane 3) compared to unmodified protein (lane 1). (D) RasG12V protein incubated in the presence or absence of JAK2, EGFR, or SRC proteins. Increased recognition of RasG12V protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 2), EGFR (lane 3) and SRC (lane 4) compared to unmodified protein (lane 1). RasY4F protein incubated in the presence or absence of JAK2, EGFR, or SRC proteins. No difference in recognition of RasY4F protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 2), EGFR (lane 3) and SRC (lane 4) compared to unmodified protein (lane 1). Basal recognition of RasY4F protein by anti-pY4 antibodies is decreased compared to RasWT protein. (E-H) Full gels of gel slices from Fig 5B. (E) RasWT protein incubated in the presence or absence of JAK2, EGFR, or SRC proteins. Increased recognition of RasWT protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5) and SRC (lane 7) but not EGFR (lane 6) compared to unmodified protein (lane 1). (F) RasG12V protein incubated in the presence or absence of JAK2, EGFR, or SRC proteins. Increased recognition of RasG12V protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5) and SRC (lane 7) but not this preparation of EGFR (lane 6) compared to unmodified protein (lane 1). (G) RasY4F protein incubated in the presence or absence of JAK2, EGFR, or SRC proteins. No difference in recognition of RasY4F protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5), EGFR (lane 6) and SRC (lane 7) compared to unmodified protein (lane 1). (H) RasY4F,G12V protein incubated in the presence or absence of JAK2, EGFR, or SRC proteins. No difference in recognition of RasY4F protein by anti-pY4 antibodies is seen upon incubation with JAK2 (lane 5), EGFR (lane 6) and SRC (lane 7) compared to unmodified protein (lane 1). Anti-pY4 antibodies also recognize JAK2 (indicated by “J” in C-H) and SRC (indicated by “S” in C-H), but this recognition does not interfere with detection of Ras proteins that run at a different size.

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(A) Control GMR-gal4/+ eye. (B) Ras G12V expressed using GMR-gal4. Eyes are rough and show some loss of eye pigment. Some eyes have black tissue at the periphery of the eye (arrow). (C) RasY4F,G12V expressed using GMR-gal4. Eyes are rough and show some loss of eye pigment. Some eyes have black tissue at the periphery of the eye (arrow). Eyes in A-C also appear in Fig 5, as these experiments were done concurrently. (D) EGFR driven by GMR-gal4 control eye. Eyes are rough and show some loss of eye pigment. (E) EGFR expressed concurrently with RasG12V using GMR-gal4. Eyes are rough, show more dramatic loss of eye pigment throughout the eye, and show consistent black tissue in the anterior periphery of the eye (arrow). (F) EGFR expressed concurrently with RasY4F,G12V using GMR-gal4. Eyes are rough, show more dramatic loss of eye pigment throughout the eye, and show consistent black tissue in the anterior periphery of the eye (arrow). Eyes also consistently show black tissue in other regions of the eye (arrowhead). Female eyes are shown.

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Raw Data for Fig 1E–1E”.

Data tables indicating (1) the raw measurements from Image J for mono- and di-ubiquitin conjugates or unconjugated (full length) protein for RasWT, Rabex-5+RasWT, RasY4E, RasY4E, Rabex-5+RasY4E, RasY4F, and Rabex-5+RasY4F, (2) the corresponding percentage calculations graphed in Fig 1E’, and (3) calculations of the relative Rabex-5 mediated ubiquitination (normalizing the calculations to the percent ubiquitination of Rabex-5+RasWT) graphed in Fig 1E”.

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Raw data for Fig 2G.

Raw measurements (pixel counts of wing tracings) for homozygous ms1096-gal4 control wings, wings homozygous for ms1096-gal4 and UAS RasWT, and wings homozygous for ms1096-gal4 and UAS RasY4F. Calculations of the average area for each set of wings are shown. Also shown are calculations to give the normalized wing areas (raw wing size divided by the average wing area for ms1096-gal4 control wings), and averages of normalized wing areas graphed in Fig 2G.

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Raw data for Fig 5I.

Raw data reporting the number of eyes with no black tissue or eyes with black tissue for eyes in which GMR-gal4 is driving expression of RasG12V, RasG12V concurrent to hop RNAi, RasY4F,G12V, and RasY4F,G12V concurrent to hop RNA. The recorded data and percentage calculations are shown.

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K-Ras, N-Ras, and H- Ras ubiquitination sites.

Table summarizing reported sites of lysine (K) ubiquitination of mouse and human K-Ras, N-Ras, and H-Ras, their reported biological roles, and their conservation in Drosophila Ras.

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