CALMODULIN-BINDING TO AND CAMP-DEPENDENT PHOSPHORYLATION OF KINESIN LIGHT-CHAINS MODULATE KINESIN ATPASE ACTIVITY

Kinesin is an ubiquitous heterotetrameric microtubule-based motor whic h translocates membrane-bound organelles. Since organelle motility and motor protein function can be regulated by components of signaling pa thways, the ability of purified bovine brain kinesin (kinesin) to be p hosphorylated and to recognize calmodulin (CaM) was tested. Extensivel y purified ''kinesin'' was found to consist of several forms of both h eavy (KHC) and light (KLC) chains. Phosphorylation of kinesin by a var iety of protein kinases was examined; cAMP-dependent protein kinase (c AMP-PK) was the most active enzyme leading to the incorporation of up to 8 mol P/mol kinesin. Phosphorylation occurred predominantly on the KLCs and led to substantial acidic pl shifts. Peptide maps indicated t hat multiple phosphorylation sites exist on each KLC. Incubation of ki nesin in vitro with protein kinase C (PKC) led to the phosphorylation of both KHCs and KLCs. In vivo phosphorylation of KHC and KLCs was dem onstrated by immunoprecipitation of [P-32]-labeled kinesin from cultur ed rat hippocampal pyramidal neurons; kinesin phosphorylation was stim ulated by 8-chlorophenylthio-cAMP or 12-O-tetradecanoylphorbol-13-acet ate. Native bovine brain kinesin was shown to bind I-125-CaM by nucleo tide-dependent pelleting with stable microtubules. Specific calcium-de pendent binding of I-125-CaM to KLCs but not KHC was found using a lig and blotting assay. cAMP-PK phosphorylated kinesin bound I-125-CaM les s well than untreated protein in both ligand blotting and microtubule- pelleting paradigms. Calcium-dependent binding of CaM to kinesin inhib ited the ATPase activity of native kinesin but not of cAMP-PK phosphor ylated kinesin. These results suggest that the KLCs have a regulatory function and integrate information coming from diverse signaling pathw ays to modulate the activity and function of kinesin.