Title: Pak1 Phosphorylation on T212 Affects Microtubules in Cells Undergoing Mitosis
Abstract: The Pak kinases are targets of the Rho GTPases Rac and Cdc42, which regulate cell shape and motility [1Sells M.A Boyd J.T Chernoff J p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts.J. Cell Biol. 1999; 145: 837-849Crossref PubMed Scopus (323) Google Scholar, 2Bagrodia S Cerione R.A Pak to the future.Trends Cell Biol. 1999; 9: 350-355Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 3Manser E Lim L Roles of PAK family kinases.Prog. Mol. Subcell. Biol. 1999; 22: 115-133Crossref PubMed Scopus (57) Google Scholar, 4Daniels R.H Bokoch G.M p21-activated protein kinase a crucial component of morphological signaling?.Trends Biochem. Sci. 1999; 24: 350-355Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 5Symons M Adhesion signaling PAK meets Rac on solid ground.Curr. Biol. 2000; 10: R535-R537Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar]. It is increasingly apparent that part of this function is due to the effect Pak kinases have on microtubule organization and dynamics. Recently, overexpression of Xenopus Pak5 was shown to enhance microtubule stabilization, and it was shown that mammalian Pak1 may inhibit a microtubule-destabilizing protein, Op18/Stathmin [6Cau J Faure S Comps M Delsert C Morin N A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization.J. Cell Biol. 2001; 155: 1029-1042Crossref PubMed Scopus (63) Google Scholar, 7Daub H Gevaert K Vandekerckhove J Sobel A Hall A Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16.J. Biol. Chem. 2001; 276: 1677-1680Crossref PubMed Scopus (230) Google Scholar]. We have identified a specific phosphorylation site on mammalian Pak1, T212, which is targeted by the neuronal p35/Cdk5 kinase [8Nikolic M Chou M.M Lu W Mayer B.J Tsai L.H The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity.Nature. 1998; 395: 194-198Crossref PubMed Scopus (342) Google Scholar, 9Rashid T Banerjee M Nikolic M Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology.J. Biol. Chem. 2001; 276: 49043-49052Crossref PubMed Scopus (123) Google Scholar]. Pak1 phosphorylated on T212, Pak1T212(PO4), is enriched in axonal growth cones and colocalizes with small peripheral bundles of microtubules. Cortical neurons overexpressing a Pak1A212 mutant display a tangled neurite morphology, which suggests that the microtubule cytoskeleton is affected [9Rashid T Banerjee M Nikolic M Phosphorylation of Pak1 by the p35/Cdk5 kinase affects neuronal morphology.J. Biol. Chem. 2001; 276: 49043-49052Crossref PubMed Scopus (123) Google Scholar]. Here, we show that cyclin B1/Cdc2 phosphorylates Pak1 in cells undergoing mitosis. In the developing cortex and in cultured fibroblasts, Pak1T212(PO4) is enriched in microtubule-organizing centers and along parts of the spindles. In living cells, a peptide mimicking phosphorylated T212 accumulates at the centrosomes and spindles and causes an increased length of astral microtubules during metaphase or following nocodazole washout. Together these results suggest that similar signaling pathways regulate microtubule dynamics in a remodeling axonal growth cone and during cell division.