Dephosphorylation of Threonine 169 of Cdc28 Is Not Required for Exit from Mitosis but May Be Necessary for Start in Saccharomyces cerevisiae

Abstract

Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions. Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G 1 cyclin Cln2. Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G 1 cyclins. We propose that in S. cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle.

Molecular Evolution Allows Bypass of the Requirement for Activation Loop Phosphorylation of the Cdc28 Cyclin-Dependent Kinase

“…Although negatively charged residues substitute effectively for phosphorylated residues in some proteins (for example, in the Cdk7 activation loop [29]) the substitution of glutamic acid for the site of activation loop phosphorylation (T169) in the Cdk Cdc28p results in very low protein kinase and biological activity (24). Previous studies with this mutant (27) were limited by the inability of the mutant to function as the sole CDC28 gene in the cell. We therefore performed error-prone PCR amplification of cdc28-T169E, and identified CDC28 plasmids that retained the T169E mutation but rescued viability in a cdc28::HIS3 deletion strain ( Fig.…”

“…2), they were surprisingly defective in Cln2p-associated kinase. Cdc28p-4324 bound to Cln2p with about 50% wild-type efficiency, but gave only 2 to 3% of wild-type Cln2p-associated kinase activity; Ϫ4321 and Ϫ5331 were about as defective as Cdc28p-T169E (27) at Cln2p binding and Cln2p-associated kinase (Fig. 3C).…”

“…In the budding yeast Cdk Cdc28p, the same mutation (T169E) resulted in a low level of Cdc28p kinase activity, correlated with a limited ability to complement the cdc28-1N allele (an allele with a G 2 -M-phase-specific defect) and no ability to complement the G 1 -S and G 2 -M-defective cdc28-4 allele (27). Although the inactivity of cdc28-T169E could indicate a requirement for a cycle of T169 phosphorylation and dephosphorylation for cell cycle control (27), the glutamic acid substitution might simply be unable to fully substitute chemically for phosphothreonine in supporting active kinase architecture (20).…”

Isolation and Characterization of New Alleles of the Cyclin-Dependent Kinase Gene CDC28 with Cyclin-Specific Functional and Biochemical Defects

“…The cdc28-T169A allele has been shown to be inactive as a kinase yet able to associate with both Cln2 and Clb2 proteins (20). cdc28-T169A was unable to rescue the pheromone sensitivity of this strain (data not shown).…”

T-Loop Phosphorylation ofArabidopsisCDKA;1 Is Required for Its Function and Can Be Partially Substituted by an Aspartate Residue

“…Phosphorylation in the T-loop has been found to be required for CDK function in organisms from three eukaryotic kingdoms-animals, yeast, and now plants-and thus appears to be a unifying principle of CDK regulation and action in eukaryotes. However, a phospho-mimicry substitution can complement a cdc2/cdc28 mutant only in plants, whereas similar phospho-mimicry substitutions did not rescue the respective mutants in budding or fission yeast (Lim et al, 1996;Gould et al, 1998). This difference between Arabidopsis and yeast becomes most obvious in the complete rescue of the gametophytic (C) and (D) PI (red) and DAPI (white) stains were used to visualize meiotic cells and DNA, respectively.…”