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B Battelle, KE Kempler, R Yamashita, JR Sellers; A Photoreceptor-specific Unconventional Myosin Is a Protein Kinase . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1385.
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Purpose: To test whether a photoreceptor-specific unconventional myosin (myosin III) is a protein kinase. Myosin III (MyoIII) is found in the photoreceptors of Limulus and Drosophila. cDNAs encoding myosin IIIs have also been cloned from vertebrate retinas (fish and human). Thus, this protein is probably important for photoreceptor and/or retinal function. Limulus MyoIII is a major target for PKA-stimulated phosphorylation in response to a circadian efferent neural input that dramatically increases photoreceptor sensitivity and responsiveness to light. MyoIII may mediate some aspects of this enhanced photoreceptor function; thus we have initiated a detailed study of its biochemistry and function. MyoIIIs have a kinase domain at their N-terminus. Here we tested whether Limulus MyoIII is a protein kinase. Methods: Full-length recombinant (FLAG-tagged) MyoIII (rMyoIII) was co-expressed with calmodulin, its presumptive light chain, in sf9 cells and purified with calmodulin as a soluble protein by FLAG affinity chromatography. rMyoIII was incubated with P ATP under standard and modified phosphorylation conditions to test whether the protein autophosphorylates. We also tested whether rMyoIII was a substrate for PKA. The incorporation of P into rMyoIII separated by SDS PAGE was quantified by phosphoimage analysis. Results: rMyoIII autophosphorylates on serine residues. The rate of autophosphorylation was linear with time for at least 3 hr and with protein concentrations between 0.04 and 4uM. Increasing the ATP concentration in the reaction mixture from 10 to 100 uM did not increase the rate of autophosphorylation. rMyoIII became heavily phosphorylated in the presence of PKA. Conclusion: Recombinant Limulus MyoIII can be expressed as a full-length soluble protein that exhibits serine/threonine kinase activity and autophosphorylates. Autophosphorylation is probably an intramolecular event. rMyoIII is also a substrate for PKA, as is the endogenous protein. The availability of large quantities of soluble, active rMyoIII will facilitate studies of its function in photoreceptors.
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