Cell elongation and branching are regulated by differential phosphorylation states of the nuclear Dbf2‐related kinase COT1 in Neurospora crassa

Abstract

SummaryDysfunction of the Neurospora crassa nuclear Dbf2-related kinase COT1 leads to cessation of tip extension and massive induction of new sites of growth. To determine the role phosphorylation plays in COT1 function, we mutated COT1 residues corresponding to positions of highly conserved nuclear Dbf2-related phosphorylation sites. Analyses of the point-mutation cot-1 strains (mimicking non-and constitutively phosphorylated states) indicate the involvement of COT1 phosphorylation in the regulation of hyphal elongation and branching as well as asexual development by altering cell wall integrity and actin organization. Phosphorylation of COT1's activation segment (at Ser417) is required for proper in vitro kinase activity, but has only a limited effect on hyphal growth. In marked contrast, even though phosphorylation of the C-terminal hydrophobic motif (at Thr589) is crucial for all COT1 functions in vivo, the lack of Thr589 phosphorylation did not significantly affect in vitro COT1 kinase activity. Nevertheless, its regulatory role has been made evident by the significant increase observed in COT1 kinase activity when this residue was substituted in a manner mimicking constitutive phosphorylation. We conclude that COT1 regulates elongation and branching in an independent manner, which is determined by its phosphorylation state.

Mitotic Exit Control of the Saccharomyces cerevisiae Ndr/LATS Kinase Cbk1 Regulates Daughter Cell Separation after Cytokinesis

“…We and others have shown that the in vivo function of Cbk1 and closely related kinases requires phosphorylation at the HM site (29,45,71), consistent with the broad importance of this regulatory mechanism among AGC kinases. However, the role this phosphorylation plays in the cell is unclear.…”

“…Like other AGC family kinases, Cbk1 is controlled by two key phosphorylations: one in the kinase activation loop (Tloop), and another within a highly conserved C-terminal hydrophobic motif (the HM site, alternatively referred to as the CT motif) (29,45,52,60,71). Cbk1's T-loop modification, by intramolecular autophosphorylation, strongly activates the kinase but is only partially required for function.…”

The Regulation of Filamentous Growth in Yeast

“…In Neurospora crassa, hyphal cells are multinucleate (Ramos- Garcia et al 2009) and grow in bifurcating branches (Ziv et al 2009) that can undergo cell-to-cell fusion (Steinberg 2007;Aldabbous et al 2010). Fusion is a dynamic process that occurs by hyphal-cell recognition through a MAPK-dependent sensing mechanism (Fleissner et al 2009).…”

The NDR Kinase DBF-2 Is Involved in Regulation of Mitosis, Conidial Development, and Glycogen Metabolism in Neurospora crassa

“…COT-1 has been localized to several intracellular compartments, including the cytoplasm and nucleus, and in association with the plasma membrane and various proteins (18,19,54). The role that COT-1 plays in the formation of branched cellular structures as well as its cellular localization has been suggested to be preserved throughout evolution (54,77). Similar conservation may also exist among DBF-2 and its orthologs, of which human LATS1 and Drosophila melanogaster WTS/LATS are, by far, the most extensively analyzed (13,45,53,74,75).…”