Protein-tyrosine kinase Pyk2 mediates endothelin-induced p38 MAPK activation in glomerular mesangial cells.
Endothelin-1 (ET-1), a member of a family of 21 amino acid peptides possessing vasoconstrictor properties, is known to stimulate mesangial cell proliferation. In this study, ET-1 (100 nm) induced a rapid activation of p21(ras) in human glomerular mesangial cells (HMC). Inhibition of Src family tyrosine kinase activation with [4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or chelation of intracellular free calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester significantly decreased ET-1dependent p21(ras) activation and suggested the involvement of the cytoplasmic proline-rich tyrosine kinase Pyk2. We have observed that Pyk2 was expressed in HMC and was tyrosine-phosphorylated within 5 min of ET-1 treatment. ET-1-induced activation of Pyk2 was further confirmed using phospho-specific anti-Pyk2 antibodies. Surprisingly, Src kinase activity was required upstream of ET-1-induced autophosphorylation of Pyk2. To determine whether Pyk2 autophosphorylation mediated ET-1-dependent p21(ras) activation, adenovirus-mediated transfer was employed to express a dominant-negative form of Pyk2 (CRNK). CRNK expression inhibited ET-1-induced endogenous Pyk2 autophosphorylation, but did not abolish ET-1-mediated increases in GTP-bound p21(ras) levels. ET-1-induced activation of the p38 MAPK (but not ERK) pathway was inhibited in HMC and in rat glomerular mesangial cells expressing the dominant-negative form of Pyk2. These findings suggest that the engagement of Pyk2 is important for ET-1-mediated p38 MAPK activation and hence the biological effect of this peptide in mesangial cells.
Department of Medicine, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. Sorokin@mcw.edu
A
A
Sorokin
P
P
Kozlowski
L
L
Graves
A
A
Philip
D000256:Adenoviridae; D002461:Cell Line, Transformed; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2; D017930:Genes, Reporter; D005822:Genetic Vectors; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases / Q000378:metabolism*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D002614:Chelating Agents; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D011994:Recombinant Proteins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)