ROLE OF CALCIUM-SENSITIVE TYROSINE KINASE PYK2 CAK-BETA/RAFTK IN ANGIOTENSIN-II-INDUCED RAS/ERK SIGNALING/

In cardiac fibroblasts, angiotensin II (Ang II) induced a rapid increa se in extracellular signal-regulated kinase (ERK) activity in a pertus sis toxin-insensitive manner. This ERK activation was abolished by the G(q)-associated phospholipase C inhibitor U73122 but was insensitive to protein kinase C (PKC) inhibitors or PKC downregulation by phorbol ester. Intracellular Ca2+ chelation by BAPTA-AM or TMB-X abolished Ang II-induced ERK activation, whereas treatment with EGTA or nifedipine did not affect it. Ca2+ ionophore A23187 also induced a rapid increase in ERK activity to an extent similar to that of Ang Il stimulation Ca lmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibit ors (genistein and ST638) completely blocked ERK activation by Ang II and A23187. Both Ang II and A23187 caused a rapid increase in the bind ing of GTP to p21(Ras), which was nearly abolished by genistein and ca lmidazolium. Transfection with the dominant negative mutant of Ras and the Ras inhibitor manumycin completely inhibited Ang II-induced ERK a ctivation. It was also found for the first time that cardiac fibroblas ts abundantly expressed Ca2+-sensitive tyrosine kinase Pyk2/CAK beta/R AFTK and that Ang II markedly induced its activation in a Ca2+/calmodu lin-sensitive manner. Overexpression of the dominant negative mutant o f Pyk2 significantly attenuated Ang II- or A23187-induced ERK activiti es (36% and 38% inhibition compared with that in mock-transfected cell s, respectively) and ERK tyrosine phosphorylation levels, as well as a n increase in the binding of GTP to p21(Ras). These findings demonstra te that in cardiac fibroblasts, Ang II-induced Ras/ERK activation is d ominantly regulated by G(q)-coupled Ca2+/calmodulin signaling and that Pyk2 plays an important role in the signal transmission for efficient activation of the Ang II-induced Ras/ERK pathway.