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. 2013 Oct 21;8(10):e79464. doi: 10.1371/journal.pone.0079464

Figure 4. Dual phosphorylation of DBF-2 is required for kinase activity and septum formation.

Figure 4

(A) Functional characterization of two conserved phosphorylation sites of DBF-2. The phosphomimetic DBF-2(T671E) variant complemented Δdbf-2, while substitution of Ser499 to alanine and glutamate and Thr671 to alanine did not. Cell wall and septa were labeled with Calcofluor White. (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 in vitro 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.