Abstract
Both cAMP- and calmodulin-dependent kinases are proposed regulators of microtubule function by means of their ability to phosphorylate microtubule-associated protein 2(MAP 2). A cAMP-dependent kinase/MAP 2 complex is endogenous to microtubules. In this report, we demonstrate that an endogenous calmodulin-dependent kinase that phosphorylates MAP 2 as a major substrate is also present in microtubules prepared under conditions that preserve kinase activity. This enzyme is identical to a calmodulin-dependent kinase purified previously from rat brain cytosol. A fraction containing calmodulin-dependent kinase and MAP 2 was separated from the cAMP-dependent kinase/MAP 2 complex by gel filtration chromatography of microtubule protein in high ionic strength buffer. All of the recovered calmodulin-dependent kinase activity in microtubules eluted in a single protein peak. The specific activity of the enzyme for MAP 2 was enriched 31-fold in this fraction compared to cytosol. Two-dimensional tryptic phosphopeptide mapping revealed that the endogenous cAMP- and calmodulin-dependent kinases phosphorylated distinct sites on MAP 2. These data demonstrate that both kinases are present in microtubule preparations and that they may differentially regulate MAP 2 function by phosphorylating separate sites on MAP 2.
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Selected References
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- Bennett M. K., Erondu N. E., Kennedy M. B. Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain. J Biol Chem. 1983 Oct 25;258(20):12735–12744. [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Burke B. E., DeLorenzo R. J. Ca2+- and calmodulin-stimulated endogenous phosphorylation of neurotubulin. Proc Natl Acad Sci U S A. 1981 Feb;78(2):991–995. doi: 10.1073/pnas.78.2.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carlin R. K., Grab D. J., Siekevitz P. Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density. J Cell Biol. 1981 Jun;89(3):449–455. doi: 10.1083/jcb.89.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeLorenzo R. J. Calmodulin in neurotransmitter release and synaptic function. Fed Proc. 1982 May;41(7):2265–2272. [PubMed] [Google Scholar]
- DeLorenzo R. J., Emple G. P., Glaser G. H. Regulation of the level of endogenous phosphorylation of specific brain proteins by diphenylhydantoin. J Neurochem. 1977 Jan;28(1):21–30. doi: 10.1111/j.1471-4159.1977.tb07704.x. [DOI] [PubMed] [Google Scholar]
- Deery W. J., Means A. R., Brinkley B. R. Calmodulin-microtubule association in cultured mammalian cells. J Cell Biol. 1984 Mar;98(3):904–910. doi: 10.1083/jcb.98.3.904. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldenring J. R., Gonzalez B., McGuire J. S., Jr, DeLorenzo R. J. Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins. J Biol Chem. 1983 Oct 25;258(20):12632–12640. [PubMed] [Google Scholar]
- Jameson L., Frey T., Zeeberg B., Dalldorf F., Caplow M. Inhibition of microtubule assembly by phosphorylation of microtubule-associated proteins. Biochemistry. 1980 May 27;19(11):2472–2479. doi: 10.1021/bi00552a027. [DOI] [PubMed] [Google Scholar]
- Job D., Rauch C. T., Fischer E. H., Margolis R. L. Regulation of microtubule cold stability by calmodulin-dependent and -independent phosphorylation. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3894–3898. doi: 10.1073/pnas.80.13.3894. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keith C., DiPaola M., Maxfield F. R., Shelanski M. L. Microinjection of Ca++-calmodulin causes a localized depolymerization of microtubules. J Cell Biol. 1983 Dec;97(6):1918–1924. doi: 10.1083/jcb.97.6.1918. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nishida E., Kumagai H., Ohtsuki I., Sakai H. The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. I. Effect of calcium-dependent regulator protein on the calcium sensitivity of microtubule assembly. J Biochem. 1979 May;85(5):1257–1266. [PubMed] [Google Scholar]
- O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
- Schulman H. Differential phosphorylation of MAP-2 stimulated by calcium-calmodulin and cyclic AMP. Mol Cell Biol. 1984 Jun;4(6):1175–1178. doi: 10.1128/mcb.4.6.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sobue K., Fujita M., Muramoto Y., Kakiuchi S. The calmodulin-binding protein in microtubules is tau factor. FEBS Lett. 1981 Sep 14;132(1):137–140. doi: 10.1016/0014-5793(81)80447-4. [DOI] [PubMed] [Google Scholar]
- Vallee R. B., Bloom G. S., Theurkauf W. E. Microtubule-associated proteins: subunits of the cytomatrix. J Cell Biol. 1984 Jul;99(1 Pt 2):38s–44s. doi: 10.1083/jcb.99.1.38s. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vallee R. B., Borisy G. G. The non-tubulin component of microtubule protein oligomers. Effect on self-association and hydrodynamic properties. J Biol Chem. 1978 Apr 25;253(8):2834–2845. [PubMed] [Google Scholar]
- Weingarten M. D., Suter M. M., Littman D. R., Kirschner M. W. Properties of the depolymerization products of microtubules from mammalian brain. Biochemistry. 1974 Dec 31;13(27):5529–5537. doi: 10.1021/bi00724a012. [DOI] [PubMed] [Google Scholar]
- Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Tubulin and calmodulin. Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus. J Cell Biol. 1979 Jun;81(3):624–634. doi: 10.1083/jcb.81.3.624. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto H., Fukunaga K., Tanaka E., Miyamoto E. Ca2+- and calmodulin-dependent phosphorylation of microtubule-associated protein 2 and tau factor, and inhibition of microtubule assembly. J Neurochem. 1983 Oct;41(4):1119–1125. doi: 10.1111/j.1471-4159.1983.tb09060.x. [DOI] [PubMed] [Google Scholar]