PHAGOCYTOSIS INDUCED BY THYROTROPIN IN CULTURED THYROID-CELLS IS ASSOCIATED WITH MYOSIN LIGHT-CHAIN DEPHOSPHORYLATION AND STRESS FIBER DISRUPTION

The actin/myosin II cytoskeleton and its role in phagocytosis were exa mined in primary cultures of dog thyroid cells. Two (19 and 21 kD) pho sphorylated light chains of myosin (P-MLC) were identified by two-dime nsional gel electrophoresis of antimyosin immunoprecipitates, and were associated with the Triton X-100 insoluble, F-actin cytoskeletal frac tion. Analyses of Triton-insoluble and soluble (PO4)-P-32-prelabeled p rotein fractions indicated that TSH (via cAMP) or TPA treatment of int act cells decreases the MLC phosphorylation state. Phosphoamino acid a nd tryptic peptide analyses of P-32-MLCs from basal cells showed phosp horylation primarily at threonine and serine residues; most of the [P- 32] appeared associated with a peptide containing sites typically phos phorylated by MLC kinase. Even in the presence of the agents which ind uced dephosphorylation, the phosphatase inhibitor, calyculin A, caused a severalfold increase in MLC phosphorylation at several distinct ser ine and threonine sites which was also associated with actomyosin and cell contraction. Phosphorylation of cell homogenate proteins or the c ytoskeletal fraction with [gamma-P-32]ATP indicated that Ca2+, EGTA, o r trifluoperazine (TFP) has little effect on the phosphorylation of ML C. Both fluorescent phalloidin and antimyosin staining of cells showed distinct dorsal and ventral stress fiber complexes which were disrupt ed within 30 min by TSH and cAMP; TPA appeared to cause disruption of dorsal, and rearrangement of ventral complexes. Concomitant with MLC d ephosphorylation and stress fiber disruption, TSH/cAMP, but not TPA, i nduced dorsal phagocytosis of latex beads. While stimulation of either A or C-kinase disrupts dorsal stress fibers and rearranges actomyosin , another event(s) mediated by A-kinase appears necessary for phagocyt ic activity.