Phosphorylation of telokin by cyclic nucleotide kinases and the identification of in vivo phosphorylation sites in smooth muscle

The Ca2+-independent acceleration of dephosphorylation of the regulatory li ght chain of smooth muscle myosin and relaxation of smooth muscle by teloki n are enhanced by cyclic nucleotide-activated protein kinase(s) [Wu et al, (1998) J, Biol, Chem. 273, 11362-11369]. The purpose of this study was to d etermine the in vivo site(s) and in vitro rates of telokin phosphorylation and to evaluate the possible effects of sequential phosphorylation by diffe rent kinases. The in vivo site(s) of phosphorylation of telokin were determ ined in rabbit smooth muscles of longitudinal ileum and portal vein. Follow ing stimulation of ileum with forskolin (20 mu M) the serine at position 13 was the only amino acid to exhibit increased phosphorylation. Rabbit porta l vein telokin was phosphorylated on both Ser-13 and -19 as a result of for skolin and GTP gamma S stimulation in vivo. Point mutation of Ser-13 (to Al a or Asp) abolished in vitro phosphorylation by cyclic nucleotide-dependent protein kinases. (C) 2000 Federation of European Biochemical Societies. Pu blished by Elsevier Science B.V, All rights reserved.