Erk1/2- and p38 MAP kinase-dependent phosphorylation and activation of cPLA2by m3 and m2 receptors

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Abstract
This study examined the upstream signaling pathways initiated by muscarinic m2 and m3 receptors that mediate sustained ERK1/2- and p38 MAP kinase-dependent phosphorylation and activation of the 85-kDa cytosolic phospholipase (cPL)A2 in smooth muscle. The pathway initiated by m2 receptors involved sequential activation of Gβγi3, phosphatidylinositol (PI)3-kinase, Cdc42, and Rac1, p21-activated kinase (PAK1), p38 mitogen-activated protein (MAP) kinase, and cPLA2, and phosphorylation of cPLA2 at Ser505. cPLA2 activity was inhibited to the same extent (61 ± 5 to 72 ± 4%) by the m2 antagonist methoctramine, Gβ antibody, pertussis toxin, the PI3-kinase inhibitor LY 294002, PAK1 antibody, the p38 MAP kinase inhibitor SB-203580, and a Cdc42/Rac1 GEF (Vav2) antibody and by coexpression of dominant-negative Cdc42 and Rac1 mutants. The pathway initiated by m3 receptors involved sequential activation of Gαq, PLC-β1, PKC, ERK1/2, and cPLA2, and phosphorylation of cPLA2 at Ser505. cPLA2 activity was inhibited to the same extent (35 ± 3 to 41 ± 5%) by the m3 antagonist 4-diphenylacetoxy-N-methylpiperdine (4-DAMP), the phosphoinositide hydrolysis inhibitor U-73122, the PKC inhibitor bisindolylmaleimide, and the ERK1/2 inhibitor PD 98059. cPLA2 activity was not affected in cells coexpressing dominant-negative RhoA and PLC-δ1 mutants, implying that PKC was not derived from phosphatidylcholine hydrolysis. The effects of ERK1/2 and p38 MAP kinase on cPLA2 activity were additive and accounted fully for activation and phosphorylation of cPLA2.