Dual phosphorylation of DBF-2 is required for kinase activity and septum formation.

Abstract

<p>(<b>A</b>) Functional characterization of two conserved phosphorylation sites of DBF-2. The phosphomimetic DBF-2(T671E) variant complemented Δ<i>dbf-2</i>, while substitution of Ser499 to alanine and glutamate and Thr671 to alanine did not. Cell wall and septa were labeled with Calcofluor White. (<b>B</b>) Kinase activity and MOB-1 interaction pattern of the indicated DBF-2 variants. Hydrophobic motif phosphorylation of Thr671 was required for maximal kinase activity, while either modification of Ser499 within the activation segment reduced DBF-2 activity to ca. 30% of the wild type DBF-2 control. Phospho-site double mutant analysis indicated that substitution of Thr671 to glutamate in a S499A and S499E background could only partly restore kinase activity. Precipitated DBF-2 variants were assayed <i>in </i><i>vitro</i> using the synthetic NDR kinase peptide (KKRNRRLSVA) as substrate (n = 5). Western Blot analysis indicated equal precipitation of the co-activator protein MOB-1 with DBF-2 activation segment and hydrophobic motif variants. </p