A Calcium-dependent Tyrosine Kinase Splice Variant in Human Monocytes
ACTIVATION BY A TWO-STAGE PROCESS INVOLVING ADHERENCE AND A SUBSEQUENT INTRACELLULAR SIGNAL*
- From the ‡University of North Carolina Lineberger Comprehensive Cancer Center, the ¶Departments of Obstetrics and Gynecology and Microbiology and Immunology, and the ‖Departments of Medicine and Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Abstract
Freshly isolated human monocytes do not express p125FAK but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKβ, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125FAK, CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.
Footnotes
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↵* This work was supported in part by grants from the American Cancer Society (to H. S. E.) and the National Institutes of Health (to J. S. H.).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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↵§ These two authors contributed equally to this project.
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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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↵1 The abbreviations used are: CADTK, calcium-dependent tyrosine kinase; Ang II, angiotensin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TPA, 12-O-tetradecanoyl- phorbol-13-acetate.
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↵2 A. Villette and H. S. Earp, unpublished results.
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- Received February 5, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
