Hydrophobic motif phosphorylation coordinates activity and polar localization of the Neurospora crassa nuclear Dbf2-related kinase COT1.

Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as a conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2.

Tales of RAM and MOR: NDR kinase signaling in fungal morphogenesis.

NDR kinases are crucial for growth, differentiation, and pathogenicity in all analyzed fungal species. They require association with MOB co-activators and several scaffolding proteins for their function. Phosphorylation of two conserved residues in the activation segment and the hydrophobic motif controls the transition between an enzymatic inactive, basal active, and fully active state of the NDR kinase. Although cellular functions of NDR kinases are only beginning to emerge, regulation of small G-proteins of the Rho and Rab families and combinatorial transcriptional and translational regulation of gene expression are conserved signaling patterns among fungal and higher eukaryotic NDR kinase pathways.

The Neurospora peptide:N-glycanase ortholog PNG1 is essential for cell polarity despite its lack of enzymatic activity.

Secretory proteins are subjected to a stringent endoplasmic reticulum-based quality control system that distinguishes aberrant from correctly folded proteins. The cytoplasmic peptide:N-glycanase cleaves oligosaccharides from misfolded glycoproteins and prepares them for degradation by the 26 S proteasome. In contrast to abundant in vitro data on its enzymatic function, the in vivo relevance of peptide:N-glycanase activity remains unclear. Here we show that the PNG1 ortholog from the filamentous ascomycete Neurospora crassa is an essential protein, and its deletion results in strong polarity defects. PNG1 and its predicted binding partner RAD23 have distinct functions in N. crassa and are involved in cell wall integrity and DNA repair, respectively. Moreover, wild type PNG1 has substitutions in essential catalytic amino acids, and its deglycosylation activity is lost. These substitutions are conserved in many PNG1 orthologs of the fungal kingdom, implying a so far unrecognized enzyme-independent function of PNG1 that may only become apparent in highly polar cells such as fungal hyphae.

Two NDR kinase-MOB complexes function as distinct modules during septum formation and tip extension in Neurospora crassa.

NDR kinases are important for growth and differentiation and require interaction with MOB proteins for activity and function. We characterized the NDR kinases and MOB activators in Neurospora crassa and identified two NDR kinases (COT1 and DBF2) and four MOB proteins (MOB1, MOB2A, MOB2B and MOB3/phocein) that form two functional NDR-MOB protein complexes. The MOB1-DBF2 complex is not only essential for septum formation in vegetative cells and during conidiation, but also functions during sexual fruiting body development and ascosporogenesis. The two MOB2-type proteins interact with both COT1 isoforms and control polar tip extension and branching by regulating COT1 activity. The conserved region directly preceding the kinase domain of COT1 is sufficient for the formation of COT1-MOB2 heterodimers, but also for kinase homodimerization. An additional N-terminal extension that is poorly conserved, but present in most fungal NDR kinases, is required for further stabilization of both types of interactions and for stimulating COT1 activity. COT1 lacking this region is degraded in a mob-2 background. We propose a specific role of MOB3/phocein during vegetative cell fusion, fruiting body development and ascosporogenesis that is unrelated to the three other MOB proteins and NDR kinase signalling.

The nuclear Dbf2-related kinase COT1 and the mitogen-activated protein kinases MAK1 and MAK2 genetically interact to regulate filamentous growth, hyphal fusion and sexual development in Neurospora crassa.

Ndr kinases, such as Neurospora crassa COT1, are important for cell differentiation and polar morphogenesis, yet their input signals as well as their integration into a cellular signaling context are still elusive. Here, we identify the cot-1 suppressor gul-4 as mak-2 and show that mutants of the gul-4/mak-2 mitogen-activated protein (MAP) kinase pathway suppress cot-1 phenotypes along with a concomitant reduction in protein kinase A (PKA) activity. Furthermore, mak-2 pathway defects are partially overcome in a cot-1 background and are associated with increased MAK1 MAPK signaling. A comparative characterization of N. crassa MAPKs revealed that they act as three distinct modules during vegetative growth and asexual development. In addition, common functions of MAK1 and MAK2 signaling during maintenance of cell-wall integrity distinguished the two ERK-type pathways from the p38-type OS2 osmosensing pathway. In contrast to separate functions during vegetative growth, the concerted activity of the three MAPK pathways is essential for cell fusion and for the subsequent formation of multicellular structures that are required for sexual development. Taken together, our data indicate a functional link between COT1 and MAPK signaling in regulating filamentous growth, hyphal fusion, and sexual development.

Protein kinase Cepsilon may act as EGF-inducible scaffold protein for phospholipase Cgamma1.

Phospholipase Cgamma1 (PLCgamma1) represents a major downstream signalling component of the epidermal growth factor (EGF) receptor (EGFR) and is activated by tyrosine phosphorylation. Here we show for the first time that cellular knockdown of protein kinase Cepsilon (PKCepsilon) leads to decreased activation of PLCgamma1 by EGF and that EGF induces tyrosine phosphorylation of PKCepsilon as well as association of PKCepsilon with both EGFR and PLCgamma1. Using several mutants, co-immunoprecipitation and phosphopeptide-based pull-down experiments we found that in dependency on c-Src and EGF-stimulation PKCepsilon may bind to the c-Src-specific phosphorylation site pY845-EGFR. Furthermore, we identified a single tyrosine residue, PKCepsilon-Y573, within a consensus binding sequence of the C-terminal SH2 domain of PLCgamma1 which is critical for both tyrosine phosphorylation of PKCepsilon and its association with PLCgamma1. Thus, in particular cells and independent of the kinase activity PKCepsilon may form a signalling module with EGFR and PLCgamma1. Thereby the tyrosine phosphorylation of PLCgamma1 via the EGFR may be facilitated. This is a novel function of PKCepsilon upstream of PLCgamma1 and a novel paradigm for the EGF-induced formation of multi-protein complexes.