Phosphorylation of caldesmon by p21-activated kinase -: Implications for the Ca2+ sensitivity of smooth muscle contraction

We have previously shown that pal-activated kinase, PAK, induces Ca2+-independent contraction of Triton-skinned smooth muscle with concomitant increase in phosphorylation of caldesmon and desmin but not myosin-regulatory light chain (Van Eyk, J, E,, Arrell, D, K,, Foster, D, B., Strauss, J, D,, Heinonen, T, Y,, Furmaniak-Kazmierczak, E,, Cote, G. P,, and Mak, A. S, (1998) J, Biol, Chem. 273, 23433-23439), In this study, we provide biochemical evidence implicating a role for PAK in Ca2+-independent contraction of smooth muscle via phosphorylation of caldesmon, Mass spectroscopy data show that stoichiometric phosphorylation occurs at Ser(657) and Ser(687) abutting the calmodulin-binding sites A and B of chicken gizzard caldesmon, respectively. Phosphorylation of Ser(657) and Ser(687) has an important functional impact on caldesmon, PAK-phosphorylation reduces binding of caldesmon to calmodulin by about 10-fold whereas binding of calmodulin to caldesmon partially inhibits PAK phosphorylation, Phosphorylated caldesmon displays a modest reduction in affinity for actin-tropomyosin but is significantly less effective in inhibiting actin-activated S1 ATPase activity in the presence of tropomyosin, We conclude that PAK-phosphorylation of caldesmon at the calmodulin-binding sites modulates caldesmon inhibition of actin-myosin ATPase activity and may, in concert with the actions of Rho-kinase, contribute to the regulation of Ca2+ sensitivity of smooth muscle contraction.