Altering Neurospora Crassa MOB2A exposes specific functions and affects its interaction with the NDR kinase COT1
MOB (MPS-1 binding) proteins act as activating subunits which are required for NDR kinase function. In N. crassa, MOB2A and MOB2B have been shown to have overlapping functions. Both MOB2 proteins physically and genetically interact with COT1, a Ser/Thr kinase that is involved in the regulation of hyphal polarity and branching. Phosphorylation has been suggested to play a role in the regulation of MOB function. Two Tyr residues (Tyr117 and Tyr119) of MOB2A can potentially undergo phosphorylation. These residues were altered by site directed mutagenesis to produce mutants harboring two Phe or Glu residues (mimicking the putative unphosphorylated or constantly phosphorylated MOB2A forms, respectively). mob-2a(Y117E,Y119E) in a ∆mob-2b background is a temperature-sensitive mutant that exhibited extremely slow growth at 34oC (optimal for the wild type). When cultured at a semi-restrictive temperature (32oC) a significant reduction in growth rate (75%) and in distance between branches (50%) was observed. We have also determined that MOB2 proteins negatively regulate conidial germination and that even though MOB2A and MOB2B have some overlapping functions, MOB2B cannot compensate for MOB2A’s role in conidiation and germination. In addition to their role in a-sexual development, MOB2A/B are also involved in the sexual reproductive cycle as mutating MOB2A/B affected perithecial development. Even though Tyr117 and Tyr119 do not reside within the predicted MOB2A-COT1 physical interface (based on the yeast model), altering these residues also affected the genetic and physical interactions between MOB2A and COT1, as determined by yeast two hybrid analyses.