The Related Adhesion Focal Tyrosine Kinase Differentially Phosphorylates p130Cas and the Cas-like Protein, p105HEF1*
- Anne Astier‡§,
- Serge N. Mani釧,
- Hava Avraham‡¶,
- Hisamaru Hirai‖,
- Susan F. Law**,
- Yuzhu Zhang**,
- Erica A. Golemis**,
- Yigong Fu¶,
- Brian J. Druker‡,
- Nilou Haghayeghi§,
- Arnold S. Freedman‡§¶¶ and
- Shalom Avraham‡¶
- From the ‡Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, ¶Division of Experimental Medicine and Hematology/Oncology Research, Beth Israel-Deaconess Medical Center, Boston, Massachusetts 02115, ‖Third Department of Internal Medicine, University of Tokyo, Hongo, Tokyo 113, Japan,**Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, ‡Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland Oregon 97201, and §Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115
Abstract
The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following β1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130Cas and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130Cas and HEF1, phosphorylation of p130Cas, but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130Cas and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130Cas domains. RAFTK was able to phosphorylate the substrate domains of both p130Cas and HEF1, but only the C-terminal domain of p130Cas. Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130Cas. These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130Cas.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants CA55207 and CA66996, American Cancer Society (ACS) Grant DHP-145 (to A. S. F.), a Fellowship from the Lymphoma Foundation of America (to S. N. M.), National Institutes of Health Grants HL55445 (to S. A.) and HL51456 (to H. A.), National Institutes of Health Training Grants CA09035 (to S. F. L.) and T32 CA09035 (to Y. Z), ACS Grant CB-74749, and National Institutes of Health Grant R29-CA63366 (to E. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶¶ To whom correspondence and reprint requests should be addressed: Division of Hematologic Malignancies, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3441; Fax: 617-632-5167; E-mail: arney_freedman{at}dfci.harvard.edu.
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↵1 The abbreviations used are: RAFTK, related adhesion focal tyrosine kinase; p125FAK, FAK, focal adhesion kinase; p105HEF1, HEF1, human enhancer of filamentation 1; SH, Src homology; BCR, B cell antigen receptor; HA, hemagglutinin; RAM, rabbit anti-mouse Ig; mAb, monoclonal antibody; GST, glutathione S-transferase; SD, substrate domain; CT, C-terminal domain.
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- Received April 15, 1997.
- Revision received May 15, 1997.
