Journal of Neuroscience, Vol 15, 6222-6229, Copyright © 1995 by Society for Neuroscience
P13suc1 associates with a cdc2-like kinase in a multimeric cytoskeletal complex in squid axoplasm
M Takahashi, N Amin, P Grant and HC Pant
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
P13suc1 sepharose-conjugated beads were used to extract the kinases that
phosphorylate neurofilaments in the squid giant axon. Using Western blots
and in vitro kinase assays, we demonstrated the presence of an active
cdc2-like kinase and its putative regulators such as cyclin E, p13, and p67
in axoplasm and a P13-axoplasm complex (P13-Ax). Protein kinase A (PKA) and
casein kinase (CK) I and II were also found in the P13-Ax. Western blot
analysis of the P13-Ax also demonstrated several axonal cytoskeletal
components; e.g., neurofilaments (NFs; NF 60, 70, and 220), tubulin, actin,
and microtubule-associated proteins. NF 220 and tubulin were phosphorylated
by the kinases in the P13-Ax. To determine whether NFs bound directly to
the P13 beads, or bound indirectly by association with cdc2 kinase, a
washed, axon-derived neurofilament preparation that contained NFs, PKA,
CKl, and tubulin, but no cdc2-like kinase, yielded no bound proteins after
incubation with P13suc1. The wash supernatant from the neurofilament
preparation, however, containing the cdc2-like kinase, did yield
cytoskeletal components that bound to P13suc1. Moreover, a
bacterial-expressed cdk5 associated with P13 beads was able to complex with
selected cytoskeletal components in the washed neurofilament preparation.
These data indicate that direct binding of P13 beads with a cdc2-like
kinase could extract active multimeric complexes composed of axonal
cytoskeletal proteins and kinases. Application of P13 chromatography may be
useful in characterizing the network of functional interactions among
cytoskeletal elements and protein kinases in neurons.
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