Phosphorylation of neuronal kinesin heavy and light chains in vivo.
The microtubule-based motor protein kinesin is thought to drive anterograde organelle transport in axons, but nothing is known about how its force-generating activity or organelle-binding properties are regulated. Studies in other motility systems suggest that protein phosphorylation is a reasonable candidate for this function. I report here that the kinesin heavy chain (HC) and light chain (LC), as well as the 160-kDa kinesin-associated protein kinectin, are phosphorylated in vivo in cultures of chick sympathetic neurons and PC12 cells labeled metabolically with 32P. In neurons, both kinesin chains are phosphorylated exclusively on serine residues, and limiting tryptic digestion demonstrated that the phosphorylation sites are clustered in a region of < or = 5 kDa for the HC and < or = 14 kDa for the LC. Partial tryptic digestion of 32P-labeled HC followed by immunoblotting with SUK4 monoclonal anti-HC and fluorography showed that the sites of HC phosphorylation are outside the globular N-terminal head region where kinesin's microtubule-binding and mechanochemical activities reside. Treatment of metabolically labeled neurons with forskolin, phorbol esters, or calcium ionophore did not alter the extent of phosphorylation, the phosphoamino acid composition, or the V8 protease phosphopeptide maps of the HC, LC, and 160-kDa protein, with one exception: treatment with calcium ionophore reduced the specific activity of the LC. In addition, when kinesin from PC12 cells was compared with that from PC12-derived cell lines lacking protein kinase A activity, neither the extent of phosphorylation nor the phosphopeptide maps were altered for either chain. Phosphopeptide mapping experiments also showed that postlysis kinase activity can phosphorylate both the neuronal HC and LC at sites not phosphorylated in vivo.
Hollenbeck
P J
PJ
Department of Neurobiology, Harvard Medical School, Boston, MA 02115.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D005576:Colforsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005728:Ganglia, Sympathetic / Q000378:metabolism*; D016547:Kinesins / Q000302:isolation & purification / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014357:Trypsin
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D005576:Colforsin; D011493:Protein Kinase C; D014357:Trypsin; D016547:Kinesins; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium