TYR117 AND TYR119 ARE REQUIRED FOR PROPER MOB2A FUNCTION AND INTERACTION WITH THE NDR KINASE COT1 IN NEUROSPORA CRASSA

MOB (MPS-1 binding) proteins act as activating subunits which are required for NDR kinase function. Phosphorylation has been suggested to play a role in the regulation of MOB 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. Tyr117 and Tyr119 of MOB2A which can potentially undergo phosphorylation, 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). Either alteration resulted in a significant reduction in conidia formation, in a manner similar to that observed in the Δmob-2a mutant. However, mob-2a(Y117F,Y119F) as well as mob-2a(Y117E,Y119E) exhibited only a minor, although significant, reduction (10%) in growth rate and distance between branches, when compared to the wild type. mob-2a(Y117F,Y119F) also exhibited dense hyphal growth. These results indicate that Tyr117 and Tyr119 are important for MOB2A function. Even though Tyr117 and Tyr119 do not reside within the predicted MOB2A-COT1 interaction sites and are not predicted to be physically associated with the NDR kinase (based on the yeast model), altering these residues also affected the physical interaction between MOB2A and COT1, as determined by yeast two hybrid analyses. We conclude that in addition to its function in regulation of hyphal elongation, MOB2A is also required for conidiation in a manner which is dependent on these residues.