Interactions between Two Cytoskeleton-associated Tyrosine Kinases: Calcium-dependent Tyrosine Kinase and Focal Adhesion Tyrosine Kinase*
- From the ‡Lineberger Comprehensive Cancer Center, the Departments of ¶Pharmacology, ‖Medicine, and§Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295
Abstract
The calcium-dependent tyrosine kinase (CADTK), also known as Pyk2/RAFTK/CAKβ/FAK2, is a cytoskeleton-associated tyrosine kinase. We compared CADTK regulation with that of the highly homologous focal adhesion tyrosine kinase (FAK). First, we generated site-specific CADTK mutants. Mutation of Tyr402 eliminated autophosphorylation and significantly decreased kinase activity. Mutation of Tyr881, a putative Src kinase phosphorylation site predicted to bind Grb2, had little effect on CADTK regulation. Src family tyrosine kinases resulted in CADTK tyrosine phosphorylation even when co-expressed with the Tyr402/Tyr881 double mutant, suggesting that Src/Fyn etc. phosphorylate additional tyrosine residues. Interestingly, CADTK tyrosine-phosphorylated FAK when both were transiently expressed, but FAK did not phosphorylate CADTK. Biochemical experiments confirmed direct CADTK phosphorylation of FAK. This phosphorylation utilized tyrosine residues other than Tyr397, Tyr925, or Tyr576/Tyr577, suggesting that new SH2-binding sites might be created by CADTK-dependent FAK phosphorylation. Last, expression of the CADTK carboxyl terminus (CRNK) abolished CADTK but not FAK autophosphorylation. In contrast, FAK carboxyl terminus overexpression inhibited both FAK and CADTK autophosphorylation, suggesting that a FAK-dependent cytoskeletal function may be necessary for CADTK activation. Thus, CADTK and FAK, which both bind to some, but not necessarily the same, cytoskeletal elements, may be involved in coordinate regulation of cytoskeletal structure and signaling.
Footnotes
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↵* This work was supported in part by the American Cancer Society and National Institutes of Health (to H. S. E.), National Institutes of Health Grant GM54010 and the American Heart Association (North Carolina affiliate) (to L. M. G.), and National Institutes of Health Grant CA65910 (to W. G. C.).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 should be addressed: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Tel.: 919-966-2335; Fax: 919-966-3015; E-mail: hse{at}med.unc.edu.
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↵2 T. Harding, X. Li, L. Grave, and S. Earp, manuscript in preparation.
- Abbreviations:
- FAK
-
focal adhesion tyrosine kinase
- CADTK
-
calcium-dependent tyrosine kinase
- CRNK
-
calcium-dependent tyrosine kinase related non-kinase
- FRNK
-
focal adhesion tyrosine kinase related non-kinase
- kd
-
kinase deficient
- PAGE
-
polyacrylamide gel electrophoresis
- SH2
-
Src homology domain 2
- GST
-
glutathione S-transferase
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- Received September 15, 1998.
- Revision received December 28, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
