Changes in tyrosine-phosphorylated p190 and its association with p120 type I and p100 type II rasGAPs during myelomonocytic differentiation of human leukemic cells.

A M(r) 190,000 protein (p190) functions as a GTPase-activating protein (GAP) for Rho and Rac family proteins, which are involved in regulating cytoskeletal actin and membrane ruffling. Tyrosine-phosphorylated p190 also complexes with rasGAP, a regulator of Ras activity, thus possibly linking Ras and Rho pathways. Leukemic cells induced to differentiate along myelomonocytic lineages have increased filamentous actin (as evidenced by phalloidin staining) and extended pseudopodia, and become irregularly shaped and flattened, suggesting altered Rho and Rac function. We, therefore, hypothesized that changes in p190 and its association with rasGAP are an integral part of these shape changes. During phorbol 13-myristate 25-acetate-induced monocytic differentiation of HL60 promyelocytic and RWLeu4 chronic myelogenous leukemic cells, the total amount of p190 decreases rapidly but returns to initial levels by 12 h. In RWLeu4, this was accompanied by commensurate changes in p190 tyrosine phosphorylation and association with p120 type I rasGAP. Association of p190 and type I rasGAP was demonstrated by immunoprecipitation with antibodies to either protein. An additional band at M(r) 100,000 (p100) was detected in immunoprecipitates after 12 h of phorbol 13-myristate 25-acetate treatment. Reverse transcription-PCR and immunoblot analyses suggest that p100 is type II rasGAP, an alternatively spliced product of p120 type I rasGAP. p100 was expressed only in response to direct protein kinase C activators, but all classes of differentiation agents increased tyrosine-phosphorylated p190. Rho and Rac are known to be involved in regulating actin polymerization. The results presented here show that the association of p190 with type I rasGAP parallels increases in actin polymerization and cell adhesion. This suggests a role for p190-rasGAP interactions in phorbol 13-myristate 25-acetate-induced cytoskeletal reorganization.