v-Crk-induced cell transformation: changes in focal adhesion composition and signaling.
v-Crk is an oncogene product in which a viral Gag sequence is fused to a cellular Crk sequence. It contains one SH2 and one SH3 domain. To gain insight into the molecular mechanisms underlying v-Crk-induced cell transformation, we studied the subcellular localization and molecular interactions of v-Crk in v-Crk-transformed NIH-3T3 cells. Our results show that v-Crk specifically localizes to focal adhesions where it induces protein tyrosine phosphorylation. Subcellular fractionation studies indicated that a significant amount of v-Crk is present in the cytoskeletal cell fraction, a fraction that includes focal adhesions. Tyrosine phosphorylated proteins, including p130CAS, were also predominantly found in the cytoskeletal fraction. We show that v-Crk induces a translocation of p130CAS to the cytoskeleton, which is accompanied by hyperphosphorylation of this protein. Mutational analyses showed that functional v-Crk SH2 domain is required for the localization of v-Crk in focal adhesions. Functional v-Crk SH2 and SH3 domains were both found to be required for the observed increase in tyrosine phosphorylation of focal adhesion proteins and for the translocation and hyperphosphorylation of p130CAS. v-Crk immunoprecipitation studies revealed that cytoskeleton-associated v-Crk interacts with both p130CAS and an unidentified tyrosine kinase. These findings suggest that formation of a focal adhesion-located complex consisting of v-Crk, a tyrosine kinase and p130CAS, which may lead to the hyperphosphorylation of p130CAS. These specific and localized signaling events may represent initial steps in the process of v-Crk-induced cell transformation.
Nievers
M G
MG
Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, The Netherlands. mirjam@emsaserv.biol.ruu.nl
Birge
R B
RB
Greulich
H
H
Verkleij
A J
AJ
Hanafusa
H
H
van Bergen en Henegouwen
P M
PM
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D050719:Crk-Associated Substrate Protein; D003599:Cytoskeleton / Q000378:metabolism*; D051379:Mice; D050736:Oncogene Protein v-crk; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D018909:src Homology Domains
C493842:Bcar1 protein, mouse; D015815:Cell Adhesion Molecules; D050719:Crk-Associated Substrate Protein; D050736:Oncogene Protein v-crk; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015682:Retroviridae Proteins, Oncogenic; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases