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 possess ing vasoconstrictor properties, is known to stimulate mesangial cell prolif eration. In this study, ET-1 (100 nM) induced a rapid activation of p21(ras ) in human glomerular mesangial cells (HMC). Inhibition of Src family tyros ine 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) ethanesN,N,N',N' -tetraacetic acid acetoxymethyl ester signi ficantly decreased ET-1dependent p21(ras) activation and suggested the invo lvement of the cytoplasmic proline-rich tyrosine kinase Pyk2, We have obser ved 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 confirm ed using phospho-specific anti-Pyk2 antibodies. Surprisingly, Src kinase ac tivity was required upstream of ET-1-induced autophosphorylation of Pyk2, T o determine whether Pyk2 autophosphorylation mediated ET-1-dependent p21(ra s) activation, adenovirus-mediated transfer was employed to express a domin ant-negative form of Pyk2 (CRNK), CRNK expression inhibited ET-1-induced en dogenous Pyk2 autophosphorylation, but did not abolish ET-1-mediated increa ses 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 c ells expressing the dominant-negative form of Pyk2, These findings suggest that the engagement of Pyk2 is important for ET-1-mediated p38 MAPK activat ion and hence the biological effect of this peptide in mesangial cells.