- Short report
- Open Access
Phosphorylation of Cdc5 regulates its accumulation
© Simpson-Lavy and Brandeis; licensee BioMed Central Ltd. 2011
- Received: 15 September 2011
- Accepted: 28 December 2011
- Published: 28 December 2011
Cdc5 (polo kinase/Plk1) is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present.
Cdc5 T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans) and drastically reduces Clb2 levels.
Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.
Cdc5/polo kinase is a crucial player in cell-cycle regulation from yeast to man, and the processes and substrates it regulates have been extensively investigated. Cdc5 regulates numerous cell cycle events, including promoting the destruction of the Cdk1-Clb2 inhibitor Swe1 , CLB2 cluster transcription (including itself) , spindle-pole body separation , spindle positioning , microtubule organization , recovery from hydroxyurea , APC/C activation , mitotic exit , cytokinesis [9, 10], Cdc14 localization [11, 12] and APC/CCdh1 inhibition . However, the regulation of Cdc5 itself remains relatively uncharacterized. In response to DNA damage, Cdc5 is inhibited by Rad53 mediated phosphorylation at an unidentified site [14, 15]. Phosphorylation of T242 by Cdk1 has been reported to be essential for viability and mitotic activity , though this is disputed . Nine other sites have been reported to be phosphorylated during mitosis , of which one, T29, when mutated to alanine stabilizes Cdc5 during G1 . Four of these sites are clustered within the first 70 amino acids of Cdc5, suggesting modification of this region may be of importance to the regulation of Cdc5.
Cdc5 stability is regulated by phosphorylation of T23
The abundance of wild type Cdc5 and cdc5 T23A protein during the cell cycle was examined by treating cells with hydroxyurea (S-phase arrest), nocodazole (metaphase arrest), or rapamycin (early G1 arrest). Curiously, Cdc5 T23A protein was undetectable under all these conditions ( Figure 1B ). Examination of whole blots with anti-Cdc5 ruled out proteolytic processing ( Figure 1C ). Moreover cdc5 ΔN70 , which lacks this phosphorylation site , migrates at the expected size (Rosella Visintin, personal communication). As Cdc5 is an essential protein we assume that cdc5 T23A cells still express it, albeit at an undetectable level.
The lack of detection of this essential protein could be due to two reasons - the first is that the antibody used does not bind to this mutant. We considered this unlikely, as the antibody used is a polyclonal antibody against the entire C-terminus of Cdc5. The other possibility is that the T23A mutation is, or has, introduced a degron. To test these ideas we expressed GFP-Cdc5WT  or GFP-cdc5T23A from the Cdc5 promoter either as the sole Cdc5 or in addition to an endogenous copy of Cdc5. Surprisingly GFP-cdc5T23A was detectable, both by anti-GFP and by anti-Cdc5 ( Figure 1D ) and accumulated normally. This indicates that the T23A mutation indeed introduced a degron and that the degron was masked by the N-terminal fusion of the GFP protein.
To determine whether the instability of cdc5 T23A has an effect in trans - on other copies of Cdc5, cdc5T23A was expressed in cells containing endogenous Cdc513myc, which has reduced electrophoretic mobility. Accumulation of endogenous Cdc513myc, and of Clb2, was unaffected by the presence of cdc5T23A ( Figure 1E ), suggesting that this mutation acts only in cis.
Cdk1 is required for maintenance of Cdc5 levels in G2
The decline in Cdc5 levels was prevented by addition of cycloheximide ( Figure 3B), indicating that new proteins must be synthesized for the elimination of Cdc5 in the absence of Cdk1 activity. This Cdk1 inhibited, non-APC/CCdh1 destruction mechanism may also account for the residual instability seen in Cdc5 with both D-boxes mutated or in cdh1Δ cells , whilst Cdc5 with the first 70 amino acids deleted (cdc5 ΔN70) is completely stable in G1 [7, 12].
Phosphorylation of T23 regulates Cdc5 accumulation
APC/CCdh1 substrate proteins accumulate to maximal levels in metaphase and are either absent or present at much lower levels during the rest of the cell cycle. In cdh1 Δ cells the level of these proteins is, in most cases, uniformly high throughout the cell cycle. Strikingly this level is comparable to their metaphase level in wild type cells. This observation suggests that some additional mechanisms are likely to cap the level of these proteins, in addition to APC/CCdh1 specific degradation. In this report, we have uncovered a potential mechanism that provides a negative feedback regulation of Cdc5 levels, a toxic protein when overexpressed , enabling cells to tolerate mild overexpression of Cdc5 (D. Morgan, personal communication). This mechanism is two-fold. Firstly, Cdc5 stability in G2 is Cdk1 kinase activity dependent. In the absence of Cdk1 kinase activity, Cdc5 is degraded by a novel, APC/CCdh1 independent mechanism that requires protein synthesis and which may account for the residual instability of D-box mutated Cdc5 . Secondly, phosphorylation of T23 of Cdc5, presumably by Cdk1 , limits the rate and maximal accumulation of Cdc5, Clb2 and possibly other CLB2 cluster proteins. A potential mechanism could be via further phosphorylation of Ndd1/Fkh2 , mediated by cdc5T23E to switch off CLB2 cluster transcription when a desirable level of CLB2 cluster proteins is reached.
This research was supported by a grant from the United States-Israel Binational Science Foundation (BSF2007288), Jerusalem, Israel and by a Project Grant from the Israel Cancer Research Fund.
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