Purification of erythrocyte dematin (protein 4.9) reveals an endogenous protein kinase that modulates actin-bundling activity.
A partially purified preparation of human erythrocyte protein 4.9, consisting of 48-, 52-, and 55-kilodalton polypeptides, is capable of bundling rabbit muscle actin in vitro (Siegel, D. L., and Branton, D. (1985) J. Cell Biol. 100, 775-785). Purification schemes, peptide mapping, antibody cross-reactivity, and chemical cross-linking techniques show that the 48- and 52-kDa polypeptides are sequence-related phosphorylated components, whereas the 55-kDa polypeptide is not. Purified protein 4.9 (dematin), consisting of 48- and 52-kDa polypeptides, effectively bundles actin in vitro; under similar conditions, the isolated 55-kDa polypeptide does not bundle actin. In fact, when added back to purified dematin, fractions containing the 55-kDa polypeptide can completely abolish dematin's actin-bundling activity. The basis for this inhibitory activity is an endogenous protein kinase that phosporylates both the 48- and 52-kDa isoforms of dematin, thus abolishing dematin's actin-bundling activity (Husain-Chishti, A., Levin, A., and Branton, D. (1988) Nature 334, 718-721). Although the endogenous kinase often co-purifies with the 55-kDa polypeptide, it can be separated from the 55-kDa polypeptide and has the characteristics of a catalytic subunit of a cyclic AMP-dependent protein kinase.
Husain-Chishti
A
A
Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138.
Faquin
W
W
Wu
C C
CC
Branton
D
D
D000199:Actins / Q000378:metabolism; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000097:blood* / Q000302:isolation & purification; D013379:Substrate Specificity
D000199:Actins; D001798:Blood Proteins; D010748:Phosphopeptides; D011494:Protein Kinases