Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines.

Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function.

Introduction

The basic Helix-loop-Helix (bHLH) transcription factors HAND1 and HAND2 are members of the twist family of bHLH proteins (for review (1). HAND factors are expressed in a variety of tissues during murine development including the heart, cardiac neural crest, lateral mesoderm, extraembryonic mesoderm, maternally derived decidua and sympathetic nervous system. bHLH factors activate/repress transcription by forming dimers via their HLH motif and bind DNA through the juxtaposition of the 2 basic domains which recognize a canonical cis-element CANNTG termed an E-box (for review (2)). bHLH proteins can generally be separated into 2 classes. The class A bHLH factors (E-proteins) which are ubiquitously expressed and the tissue-restricted or class B bHLH factors. The established paradigm of function was that a heterodimer composed of a class A and a class B protein was required for transcriptional regulation and in fact many class B factors do not form homodimers or heterodimers with other class B factors efficiently. Recently, we and others recognized that members of the twist family of proteins exhibited a more promiscuous dimerization profile forming homodimers and heterodimers with a wide range of class B factors (3-6). With this in mind, we set out a hypothesis that tissue-specific transcriptional regulation by HAND factors was in fact driven by the specific bHLH dimer complex that could form. This hypothesis begs the question of how is HAND dimerization controlled. Most recently, we determined that the answer to this question is in part addressed by post-translational modification (phosphorylation) of specific residues in the bHLH domain and that these modifications affect the dimerization affinities of HAND proteins that allow for changes in biological function (3). To determine if in fact HAND factors were phosphorylated and to define the specific location of the modified residues, we coexpressed a flag epitope tagged HAND1 (FlagHAND1) with constitutively active kinases and or phosphatases in tissue culture cells (HEK293 and RCHOI), which were subsequently metabolically labeled with 32P-orthophosphate, HAND1 protein was immuno-precipitated using flag antibody, subjected to trypsin digestion, peptides were separated via 2-dimensional phosphopeptide mapping and phosphopeptides were visualized via exposure of the TLC plates to phosphoimager screens. By employing HAND1 deletion and point mutants, this technique allowed for the identification of the specific residues that are phosphorylated.

Materials and Methods

Constructs

PKC-7 is a constitutively active form of PKCα, which was previously described (7). pFC-PKA is a constitutively active PKA (Strategene). pIRES FLAG-HAND1 and HAND1 point mutants are amino FLAG-tag (Sigma) fusions cloned into pIRES NEO (Clonetech). B56α and δ cDNAs were cloned into the expression plasmid pCEP4-l. HAND1 point mutants HAND1 S98A, S109A-T107A &D were generated using the Quick-Change Mutagenesis kit (Strategene) following the manufacturers protocols.

Tissue culture

HEK293 cells were grown in 10% FBS containing DMEM containing antibiotics at 37oC and 5% CO2. A total of 10 μg of the indicated plasmid construct was transfected into cells using a CaPO4 based transfection (8). Briefly DNA was resuspended in 50 μl of H2O. To this 500 μl of 2X HBS was added. While bubbling air constantly with an autopipette, 450 μl of a 2:12 dilution of 2 M CaCl2 was added drop-wise and allowed to sit for 20 minutes at room temperature. Precipitates were then added to cells and allowed to incubate for 4 hours at 37oC and 5% CO2. Media was removed and replaced with 3 ml of 15% glycerol in supplemented DMEM for 1 minute. Media was immediately aspirated, cells were washed in 5 mL of 1X PBS and 10 ml of supplemented DMEM was added and cells were grown for 48 hours.

labeling-Immunoprecipitations

HEK293 cells were grown and transfected as described above with the indicated constructs. 48 hours post transfection cells were incubated with 1 mCi of 32P orthophosphoric acid (NEN)/ml of phosphate–free DMEM supplemented with dialyzed FBS for 4 hours. Cells were washed in 20 mM HEPES pH 7.4 and 150 mM NaCl followed by lysis in 20 mM NaPO4, 150 mM NaCl, 2 mM MgCl2, 0.1% NP40, 10% glycerol, 3 mg/μl leupeptin, 3 mg/μl pepstatin, 1 mM PMSF, 50 μM NaVO4, 5 mM NaF, 100 nM Okadaic acid, and 5 mM Beta glycerol phosphate. Equal amounts of protein were immunoprecipitated with agarose-conjugated FLAG M2-beads (Sigma) for 2 hours. Samples were washed 3 times in 1X PBS with a tube change on the last wash. Samples were boiled in loading dye, run through a 12% SDS PAGE, dried and exposed to a phosphoimager screen.

2-dimensional phosphopeptides mapping

HEK293 cells were grown, transfected, labeled with 32P, and immunoprecipitated as described above. Dried IP gels were exposed to phosphoimager. Bands were identified and cut by aligning the image with radioactive marker spots. The acrylamide bands corresponding to labeled FLAG-HAND1 were cut away from the dried gel, the filter paper was scraped away from the acrylamide, and rehydrated in 400 μl freshly made 50 mM ammonium bicarbonate. The rehydrated acrylamide was crushed into small pieces and allowed to digest overnight with 30 μg of TPCK-treated trypsin (Worthington) at 37ºC. Digests were spiked the following day. Digested peptides were removed from the crushed acrylamide, washed twice with 50 mM ammonium bicarbonate, and the peptides were concentrated in a Speed Vac (Forma). Digested peptides were washed 4 times with 1 mL ddH2O and then twice in pH 1.9 buffer (2.8% formic acid, 7.8% glacial acetic acid). Samples were then resuspended in 4 μl pH 1.9 buffer, spotted onto cellulose TLC plates and run in the 1st dimension in pH 1.9 buffer on a Hunter Thin Layer Electrophoresis apparatus (HTLE 7000, CBS Scientific, Inc.) for 35 minutes at 1300 V. Plates were dried, rotated 90 degrees, and then run in the 2nd dimension using an isobutyric acid buffer (62.5% isobutyric acid, 1.9% n-Butanol, 4.8% pyridine, 2.9% glacial acetic acid) in a TLC tank. When liquid phase migration was 1 cm from the top of the plates, they were removed, dried, and exposed to a phosphoimager screen for visualization and analysis.

Results and Discussion

We recently demonstrated that HAND1 is phosphorylated during the differentiation of RCHOI trophoblast stem cells and that protein kinase A (PKA), protein kinase C (PKC) and the delta isoform of the B56-regulatory subunit (B56δ) of the protein phosphatase 2A (PP2A) regulate HAND1 phosphorylation status on residues T107 and S109 (3). Key in this study was the ability to determine the exact residues that were being modified in HAND1 so that functional analysis of HAND1 mutants in these residues could be deduced. We employed phosphopeptide analysis to address these questions as it provided a greater sensitivity to changes in HAND1 phosphorylation state than simple immunoprecipitations (IPs) of HAND1 expressed in metabolically labeled cells. We chose to use phosphopeptide mapping over mass spectrophometry as we could perform these experiments ourselves without needing to rely on instrumentation analysis by others. Results show that expression of constitutively active PKC along with HAND1 results in increased phosphorylation of 5 peptides (Fig. 1; 3). Moreover, coexpression of B56α results in no significant change to HAND1 phosphorylation while coexpression of B56δ reduces the phosphorylation on spots 8 and 9 correlating well with B56δ interaction analysis with HAND1 (Fig. 1; 3).

Fig. 1

Identification of HAND1 residues phosphorylated by PKC using phosphopeptide analysis in HEK293 cells.

Panels (A) and (B) show the variation of HAND1 phosphorylation when expressed with or without constitutively active PKC. The increased signal intensity of 5-phosphopepties (5-9) indicates increased phosphorylation. Panels (C) and (D) show that coexpression of the non-interacting B56α (C) has no effect on HAND1 phosphorylation by PKC whereas expression of B56δ (D) reduces HAND1 phosphorylation of peptides 8 and 9 (marked *). Panel (E) shows that point mutagenesis of both T107 and S109 to alanine eliminates the phosphorylation of peptides 8 and 9 (marked X) confirming these sites as PKC targets and targets for dephosphorylation by B56δ-containing PP2A.

We next employed serine and threonine mutations based on our understanding of the trypsin restriction map of HAND1. Our designed HAND1 mutants correspond to S→A changes in consensus kinase sites for PKA and PKC. Additionally when designing the mutants, we considered if the mutations were contained within a single trypsin fragment and if other S and or T residues were also present in these peptides (3). In the case of T107 and S109, they were contained in a single fragment, so we decided to mutate these in combination. Phosphopeptide maps of HAND1 and HAND1T107;S109A coexpressed with or without constitutively active PKC show that 2-specific phosphopeptides are absent from the map of HAND1T107;S109A when compared to wildtype HAND1 (Fig. 1). These results correspond with similar previously published experiments in which we coexpressed constitutively active PKA (3). The observation that a double mutation within a single trypsin fragment resulted in the reduction of two phosphopeptides on the map can be interpreted in two ways. One is that the two spots represent mono- and diphosphorylated forms of the protein. The second is that the two peptides are in fact an artifact of incomplete trypsin digestion. Trypsin cuts at basic residues and the HAND1 basic domain contains a high concentration of basic residues. As the HAND1 basic domain is located just amino to T107 and S109 partial trypsin digestion could explain the loss of 2 phosphopeptides in the HAND1T107;S109A mutant shown in Figure 1. This phenomenon was clearly encountered in the mutation of S98 (the only S within its trypsin peptide) where 3-5 partial fragments were reduced in the maps of this HAND1 mutant (3). To try to address this issue directly, we made the single HAND1 mutants T107A and S109A, coexpressed these mutants in cells with constitutive kinase and compared maps to wild type HAND1 (Fig. 2). Results of these experiments show that mutation of T107 to alanine does not significantly alter phosphopeptide pattern of HAND1 when coexpressed with PKA; however, mutation of S109 eliminates phosphorylation of both peptides (Fig. 2). These results suggest that indeed like S98 partial digestion may come into play, but in this case it is also possible that phosphorylation of T107 is required for phosphorylation of S109. To address this question would require mass spectrophometery.

Fig. 2

Single mutations of T107 and S109 suggest that S109 is the main target of PKA and PKC.

HAND1, HAND1T107A or HAND1S109A were coexpressed with constitutively active PKA in HEK293 cells and subjected to phosphopeptide analysis. Mutation of T107 shows no significant difference in the HAND1 phosphopeptide map specifically peptides 8 and 9 (see Fig. 1). In contrast, mutation of S109 to alanine eliminates phosphopeptides 8 and 9 and recapitulates the data observed in the double mutant. This suggests that peptides 8 and 9 are partial tryptic digests and not mono- and diphosphorylated forms of the protein.

Taken together, the results obtained show that the upregulation of HAND1 phosphorylation that is observed during RCHOI differentiation (3) can be attributed to the modifications of three residues within HAND1. It should be considered that it is possible that other residues within HAND1 may in fact be phosphorylated, but our conditions of mapping are either insufficient to resolve 2 labeled peptides or the peptides run off of the TLC plate in the solvent phase. Employing different solvents for the chromatography used in the 2nd dimension can test this latter possibility. A useful and expedient way to test different solvent conditions is to use in silico mapping simulation programs such as Mobility, which can be found on the Gene Stream web site (http://www.genestream.org/). Based on your trypsin digestion pattern the program will show you which peptides will likely be present on the peptide map using a particular solvent.