p21-Activated Kinase 1 Regulates Microtubule Dynamics by Phosphorylating Tubulin Cofactor B

Abstract

p21-activated kinase 1 (Pak1) induces cytoskeleton reorganization in part by regulating microtubule dynamics through an elusive mechanism. Using a yeast two-hybrid screen, we identified tubulin cofactor B (TCoB) (a cofactor in the assembly of the α/β-tubulin heterodimers) as an interacting substrate of Pak1. Pak1 directly phosphorylated TCoB in vitro and in vivo on serines 65 and 128 and colocalized with TCoB on newly polymerized microtubules and on centrosomes. TCoB interacted with the GTPase-binding domain of Pak1 and activated Pak1 in vitro and in vivo. In contrast to wild-type TCoB, an S65A, S128A double mutant and knock-down of the endogenous TCoB or Pak1 reduced microtubule polymerization, suggesting that Pak1 phosphorylation is necessary for normal TCoB function. Overexpression of TCoB dramatically increased the number of γ-tubulin-containing microtubule-organizing centers, a phenotype reminiscent of cells overexpressing Pak1. TCoB was overexpressed and phosphorylated in breast tumors. These findings reveal a novel role for TCoB and Pak1 in regulating microtubule dynamics.

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p21-Activated Kinase 1 Regulates Microtubule Dynamics by Phosphorylating Tubulin Cofactor B

ACKNOWLEDGMENTS

This work was supported by the U.S. National Institutes of Health grants CA90970, CA80066, and P50CA97007 (to R.K.). H.V.G. was supported in part by the American Heart Association.

We thank Liana Adam for the γ-tubulin confocal microscopy and Sally A. Lewis, New York University Medical Center, for providing cofactor B pet23d vector.