Nak1, an Essential Germinal Center (GC) Kinase Regulates Cell Morphology and Growth in Schizosaccharomyces pombe

Abstract

We have identified and characterized Nak1, a 652-amino acid NH 2 -terminal kinase belonging to the group II germinal center kinase (GCK) family, in Schizosaccharomyces pombe. We found that nak1 is essential for cell proliferation. Furthermore, partial repression of nak1, under regulation of an integrated nmt1 promoter, resulted in an aberrant round cellular morphology, actin and microtubule mislocalization, slow growth, and cell division defects. Overexpression of either a kinaseinactive mutant (Nak1 K39R ) or the non-catalytic domain resulted in similar phenotypes, suggesting dominantnegative effects. By deletion analysis, we mapped the region responsible for this dominant-negative effect to the COOH-terminal 99 residues. Furthermore, we found that deletion of the COOH-terminal 99 residues inhibited Nak1 autophosphorylation, and expression of a partially inactive (Nak1 T171A ) or truncated (Nak1 1-562 ) protein only weakly suppressed morphological and growth phenotypes, indicating that both kinase and COOH-terminal regions are important for Nak1 function. GFPNak1 localized uniformly throughout the cytoplasm, unlike many other proteins which influence cell polarity that preferentially localize to cell ends. Together, our results implicate Nak1 in the regulation of cell polarity, growth, and division and suggest that the COOH-terminal end plays an important role in the regulation of this kinase.

The distinct wiring between cell cycle regulation and the widely conserved Morphogenesis-Related (MOR) pathway in the fungus Ustilago maydis determines the morphological outcome

“…This pathway includes a nuclear Dbf2-related (NDR) kinase termed Cbk1 in Saccharomyces cerevisiae (Racki et al, 2000), Orb6 in Schizosaccharomyces pombe (Verde et al, 1998), and COT1 in Neurospora crassa (Yarden et al, 1992), associated with a regulatory subunit, Mob2 (Hou et al, 2003;Maerz et al, 2009;Nelson et al, 2003). The NDR-Mob2 complex is activated by a germinal center kinase called Kic1 in S. cerevisiae (Nelson et al, 2003), Nak1 in S. pombe (Huang et al, 2003), and Pod6 in N. crassa (Seiler et al, 2006). Additional elements are Hym1/Mo25-related (Karos and Fischer, 1999;Nelson et al, 2003) and Tao3/ Mor2/Fry-related scaffold-like proteins (Du and Novick, 2002;Kanai et al, 2005).…”

Nak1 interacts with Hob1 and Wsp1 to regulate cell growth and polarity inSchizosaccharomyces pombe

“…pREP3× and pREP4× contain the strong thiamine-repressible nmt1 promoter (Forsburg, 1993). pAALNHA-Nak1 has been previously described (Huang et al, 2003). pAALNHA-Hob1, pAALNHAHob1 1-281 and pAALNHA-Hob1 275-466 were constructed by insertion of Hob1-encoding fragments into the SpeI/BamHI sites of pAALNHA.…”

“…pAALNHA contains LEU2, ars1 and the adh1 promoter sequence flanking a hemagglutinin (HA)-epitope-coding sequence and a polylinker site (Huang et al, 2003). pREP3× and pREP4× contain the strong thiamine-repressible nmt1 promoter (Forsburg, 1993).…”

“…Several Schizosaccharomyces pombe polarity mutants result in either asymmetric growth (for3, bud6, tea1, pom1) or rounded morphology (cdc42, orb2/pak1/shk1, orb5, orb6, mor2, scd1, nak1) (Miller and Johnson, 1994;Chang et al, 1994;Verde et al, 1995;Mata and Nurse, 1997;Yang et al, 1998;Glynn et al, 2001;Feierbach and Chang, 2001;Hirata et al, 2002;Huang et al, 2003). Although it is not clear how these factors mediate polarity, several of these mutants exhibit actin cytoskeleton defects.…”

“…By contrast, Sid1 mediates septation during cell division (Balasubramanian et al, 1998;Guertin et al, 2000). We previously reported that Nak1 is essential for cell growth and polarity, and that repression of nak1 expression resulted in abnormal round morphology, disorganized actin foci, slow growth and cell-division defects (Huang et al, 2003). Here, we provide evidence that Hob1 and Wsp1 are involved in determining cell polarity, and that Nak1 can form complexes with these proteins and is required for their proper localization and function at growing cell ends.…”