Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II.
The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2-SH3-SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Delta242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr(222) phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Delta255 or Crk Delta242 Extended Linker (Crk Delta242([EL])), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr(397), hyperphosphorylation of focal adhesion proteins p130(cas) and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Delta242([EL]) could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP-PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.
Zvara
A
A
Laboratory of Molecular Oncology, The Rockefeller University, New York, NY 10021, USA.
Fajardo
J E
JE
Escalante
M
M
Cotton
G
G
Muir
T
T
Kirsch
K H
KH
Birge
R B
RB
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D050719:Crk-Associated Substrate Protein; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions; D051379:Mice; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D050717:Retinoblastoma-Like Protein p130; D017384:Sequence Deletion; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics*
C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; C496051:Ptk2 protein, mouse; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases; C516739:Ptpn12 protein, mouse