Activation of a Novel Calcium-dependent Protein-tyrosine Kinase

doi:10.1074/jbc.271.47.29993

Activation of a Novel Calcium-dependent Protein-tyrosine Kinase

CORRELATION WITH c-Jun N-TERMINAL KINASE BUT NOT MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION*

From the ^‡ Lineberger Comprehensive Center, Departments of Medicine and Pharmacology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599
^§ Protein and Peptide Group, European Molecular Biology Laboratory, Heidelberg D-69012, Germany,
^¶ Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709

^∥To whom correspondence should be addressed. Tel.: 919-966-3036; Fax: 919-966-3015.

Abstract

Many G protein-coupled receptors (e.g. that of angiotensin II) activate phospholipase Cβ, initially increasing intracellular calcium and activating protein kinase C. In the WB and GN4 rat liver epithelial cell lines, agonist-induced calcium signals also stimulate tyrosine phosphorylation and subsequently increase the activity of c-Jun N-terminal kinase (JNK). We have now purified the major calcium-dependent tyrosine kinase (CADTK), and by peptide and nucleic acid sequencing identified it as a rat homologue of human PYK2. CADTK/PYK2 is most closely related to p125^FAK and both enzymes are expressed in WB and GN4 cells. Angiotensin II, which only slightly increases p125^FAK tyrosine phosphorylation in GN4 cells, substantially increased CADTK tyrosine autophosphorylation and kinase activity. Agonists for other G protein-coupled receptors (e.g. LPA), or those increasing intracellular calcium (thapsigargin), also stimulated CADTK. In comparing the two rat liver cell lines, GN4 cells exhibited ∼ 5-fold greater angiotensin II- and thapsigargin-dependent CADTK activation than WB cells. Although maximal JNK activation by stress-dependent pathways (e.g. UV and anisomycin) was equivalent in the two cell lines, calcium-dependent JNK activation was 5-fold greater in GN4, correlating with CADTK activation. In contrast to JNK, the thapsigargin-dependent calcium signal did not activate mitogen-activated protein kinase and Ang II-dependent mitogen-activated protein kinase activation was not correlated with CADTK activation. Finally, while some stress-dependent activators of the JNK pathway (NaCl and sorbitol) stimulated CADTK, others (anisomycin, UV, and TNFα) did not. In summary, cells expressing CADTK/PYK2 appear to have two alternative JNK activation pathways: one stress-activated and the other calcium-dependent.

Footnotes

↵* This work was supported by National Institutes of Health Grant DK 31683 and a grant from the American Cancer Society. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U69109[GenBank].
↵¹ The abbreviations used are:

Ang II

angiotensin II

CADTK

calcium-dependent tyrosine kinase

JNK

c-Jun N-terminal kinase

PAGE

polyacrylamide gel electrophoresis

MS

mass spectroscopy

GST

glutathione S-transferase

MAP

mitogen-activated protein

TNF

tumor necrosis factor

EGF

epidermal growth factor

LPA

L-α-lysophosphatidic acid, oleoly

CAKβ

cell adhesion kinase β

RAFTK

related adhesion focal tyrosine kinase

MEKK

mitogen-activated protein kinase/extracellular signal-regulated kinase kinase

JNKK

JNK kinase

SEK

stress-activated extracellular kinase

PAK

p21^{(CDC42)/(RAC)}-activated kinase.
↵²S. Lev, J. Schlessinger, H. Yu, X. Li, R. Dy, L. Graves, and H. S. Earp, unpublished results.
↵³Li, X., Yu, H., He, Q., Gravea, L., and Earp, H. S., unpublished results.
- Received September 4, 1996.