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B. Battelle, K.E. Kempler, G.M. Mapel; Photoreceptor-Specific Myosin III: Further Characterization of its Kinase Activity and Substrate Specificity . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2864.
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Purpose: To characterize further the kinase activity and substrate specificity of myosin III (myoIII). MyoIII is an unconventional myosin found in the photoreceptors of both vertebrates and invertebrates. MyoIIIs have a kinase domain at their N-terminus and a myosin domain at their C-terminus. The kinase domain of myoIII is most closely related to GCKs of the ste20 superfamily. Previously we reported that recombinant Limulus myoIII expressed in and purified from sf9 cells autophosphorylates. Therefore myoIII can function as a protein kinase. MyoIII is quantitatively a major kinase in Limulus photoreceptors. Here we tested the ability of myoIII to phosphorylate various exogenous substrates, and we tested the effects of various kinase inhibitors of myoIII kinase activity. Methods: Purified recombinant myoIII was incubated under phosphorylating conditions in the presence of P ATP with both peptide and protein substrates. The phosphorylation of peptides was assayed using the phosphocellulose binding assay. The phosphorylation of proteins was assayed by separating the myoIII from the protein substrates by SDS PAGE, exposing the dried gels to a phosphorimager and then quantifying the labeled bands (ImageQuant). In some experiments, known inhibitors of different protein kinases were added to the incubations to test for their ability to inhibit myoIII kinase activity. Results: MyoIII phosphorylates a peptide substrate of PKA in a time and concentration-dependent manner but not peptides that are preferred substrates for PKC, CaM PKII or casein kinase I or II. It also did not phosphorylate peptides based on the sequence of the C-terminus of Limulus arrestin, a major light-stimulated phosphoprotein in photoreceptors. The phosphorylation of the PKA substrate by myoIII, and myoIII autophosphorylation, were inhibited by a peptide inhibitor of PKA, but not by peptide inhibitors of CaM PKII or PKC. MyoIII phosphorylation of the PKA substrate and myoIII autophosphorylation were also inhibited by 1uM H89 and 1 uM KT5720, two inhibitors of the catalytic subunit of PKA. Two inhibitors of PKC either enhanced (calphostin C) or did not significantly change (RO-31-8220) the ability of myoIII to phosphorylate the PKA peptide substrate. Purified recombinant myoIII can also phosphorylate the following proteins: alpha and beta casein, phosvitin and the C-terminus of Limulus opsin. Conclusion: MyoIII from Limulus photoreceptors is a multifunctional kinase that has some properties similar to the catalytic subunit of PKA. Opsin is a potential substrate for Limulus myoIII.
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