conserved orb6 phosphorylation sites are essential for polarized cell growth in schizosaccharomyces pombe守恒orb6磷酸化网站对于极化在粟酒裂殖酵母细胞生长至关重要.pdfVIP

Conserved Orb6 Phosphorylation Sites Are Essential for Polarized Cell Growth in Schizosaccharomyces pombe Guohong Liu, Dallan Young* Departments of Biochemistry Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, Canada Abstract The Ndr-related Orb6 kinase is a key regulator of polarized cell growth in fission yeast, however the mechanism of Orb6 activation is unclear. Activation of other Ndr kinases involves both autophosphorylation and phosphorylation by an upstream kinase. Previous reports suggest that the Nak1 kinase functions upstream from Orb6. Supporting this model, we show that HA-Orb6 overexpression partially restored cell polarity in nak1 ts cells. We also demonstrated by coimmunoprecipitation and in vitro binding assays that Nak1 and Orb6 physically interact, and that the Nak1 C-terminal region is required forNak1/Orb6 complex formation in vivo. However, results from in vitro kinase assays did not show phosphorylation of recombinant Orb6 by HA-Nak1, suggesting that Orb6 activation may not involve direct phosphorylation by Nak1. To investigate the role of Orb6 phosphorylation and activity, we substituted Ala at the ATP-binding and conserved phosphorylation sites. Overexpression of kinase-dead HA-Orb6K122A in wild-type cells resulted in a loss of cell polarity, suggesting that it has a dominant-negative effect, and it failed to rescue the polarity defect of nak1 or orb6 ts mutants. Recombinant GST-Orb6S291A did not autophosphorylate in vitro suggesting that Ser291 is the primary autophosphorylation site. HA-Orb6S291A overexpression only partially rescued the orb6 polarity defect and failed to rescue the nak1 defect, suggesting that autophosphorylation is important for Orb6 function. GST-Orb6T456A autophos