Phosphorylation of Kinesin inVivo Correlates with Organelle Association and Neurite Outgrowth (*)
- § To whom correspondence should be addressed: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0881; Fax: 617-734-7557; phollenb{at}warren.med.harvard.edu
Abstract
The motor protein kinesin is implicated in organelle movement toward the plus ends of microtubules, but little is known about
its interaction with organelle membranes or about the physiological role of the phosphorylation of kinesin and its associated
protein kinectin seen in neurons in vivo (Hollenbeck, P. J. (1993) J. Neurochem. 60, 2265-2275). Here we have demonstrated that the kinesin heavy chain (KHC), light chain, and kinectin isolated from chick
brain or sympathetic neurons exist in several isoelectric forms. Metabolic labeling followed by phosphatase treatment showed
that these are phosphoisoforms, and that phosphorylation is reversible in vitro. To assess the capability of phosphorylation to regulate kinesin's state and/or activity, we performed 
P and 
S pulse-chase experiments with neuronal cultures and determined that kinesin-associated phosphate turns over 3-4 times faster
than the proteins themselves. When the phosphoisoform distributions for different kinesin pools were analyzed, it was found
that membrane-associated KHC contained predominantly the most highly phosphorylated isoform, while soluble kinesin consisted
of less phosphorylated KHC isoforms. Nerve growth factor-induced neurite outgrowth in PC12 cells was found to increase significantly
kinesin's 
P specific activity while doubling the relative abundance of the most highly phosphorylated KHC isoform. These results demonstrate
that the phosphorylation state of kinesin is closely coupled to its organelle binding and to the magnitude of organelle transport
in the cell. We propose that the phosphorylation state of kinesin and associated proteins may regulate motility via association
with organelle membranes and, specifically, that KHC phosphorylation induces membrane association.
Footnotes
-
↵* This work was supported by National Institutes of Health Grant NS27073 and the Harvard Mahoney Neuroscience Institute (KDL). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- KHC
-
kinesin heavy chain
- KLC
-
kinesin light chain
- NGF
-
nerve growth factor
- DMEM
-
Dulbecco's modified Eagles medium
- PIPES
-
1,4-piperazinediethanesulfonic acid
- KAPPs
-
kinesin-associated phosphoproteins
- IEF
-
isoelectric focussing
- PAGE
-
polyacrylamide gel electrophoresis.
-
↵2 K.-D. Lee and P. J. Hollenbeck, unpublished results.
-
- Received August 1, 1994.
- Revision received December 5, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
