ACTIVATION OF MAP KINASES AND PHOSPHORYLATION OF CALDESMON IN CANINE COLONIC SMOOTH-MUSCLE

1. Phosphorylation of caldesmon was assayed in canine colonic circular smooth muscle strips labelled with P-32 and stimulated with 10 mu M a cetylcholine. Caldesmon was isolated by two-dimensional non-equilibriu m pH gel electrophoresis. Stimulation with acetylcholine increased cal desmon phosphorylation significantly from a basal level of 0.6 +/- 0.0 7 to 1.1 +/- 0.15 mol P-i (mol caldesmon)(-1) after 2 min. 2. MAP kina se activities were measured in SDS extracts of muscle by a gel reconst itution method using myelin basic protein. Myelin basic protein kinase activities were observed at 38, 44, 50 and 57 kDa by the gel reconsti tution method. Endogenous caldesmon kinase activities were also identi fied by the gel reconstitution method at 38, 44 and 50 kDa. The 38 and 44 kDa kinases comigrated with proteins labelled by anti-ERK1 MAP lii nase antibodies on Western blots. Both 38 and 44 kDa MBP kinase activi ties increased significantly during contractions induced by 10 mu M ac etylcholine, 0.1 mu M neurokinin A and 70 mM potassium. 3. Phorbol dib utyrate (0.1 mu M) potentiated activation of MAP kinases and contracti on of depolarized muscles while producing a decrease in fura-2 fluores cence ratio. This suggests that protein liinase C activation is couple d to MAP kinase activity in colonic smooth muscle. 4. MAP kinases isol ated from muscle homogenates by Mono & chromatography were assayed usi ng the specific MAP kinase substrate peptide APRTPGGRR. Stimulation of muscles for 2 min with 10 mu M acetylcholine activated both ERK1 and ERK2 MAP kinase activities 2-fold. 5. To determine the effects of cald esmon phosphorylation by MAP kinase on the cross-bridge cycle, actin s liding velocity was measured with an in vitro motility assay, Unphosph orylated turkey gizzard caldesmon (3 mu M) significantly reduced mean sliding velocity. Phosphorylation of caldesmon with sea star ERK1 MAP kinase reversed the inhibitory effect of caldesmon on sliding velocity . The results are consistent with a protein kinase cascade being activ ated by contractile agonists in gastrointestinal smooth muscle which a ctivates ERK MAP kinases leading to phosphorylation of caldesmon. Phos phorylation of caldesmon in viveo may reverse inhibitory influences of caldesmon on cross-bridge cycling.