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Normal cell cycle progression requires the precise activation and inactivation of cyclin-dependent protein kinases (CDKs), which consist of a CDK and a cyclin subunit. A novel cell cycle regulator called Speedy/Ringo shows no sequence similarity to cyclins, yet can directly bind to and activate CDKs. Speedy/Ringo proteins, which bind to and activate Cdc2 and Cdk2 in vitro, are required for the G2 to M transition during Xenopus oocyte maturation and for normal S-phase entry in cultured human cells. We have characterized the substrate specificity and enzymatic activity of human Cdk2-Speedy/Ringo A2 in order to gain insights into the possible functions of this complex. In contrast to Cdk2-cyclin A, which has a well-defined consensus target site ((S/T)PX(K/R)) that strongly favors substrates containing a lysine at the +3 position of substrates, Cdk2-Speedy/Ringo A2 displayed a broad substrate specificity at this position. Consequently, Cdk2-Ringo/Speedy A2 phosphorylated optimal Cdk2 substrates such as histone H1 and a KSPRK peptide poorly, only approximately 0.08% as well as Cdk2-cyclin A, but non-canonical Cdk2 substrates such as a KSPRY peptide relatively well, with an efficiency of approximately 80% compared to Cdk2-cyclin A. Cdk2-Speedy/Ringo A2 also phosphorylated authentic Cdk2 substrates, such as Cdc25 proteins, which contain non-canonical CDK phosphorylation sites, nearly as well as Cdk2-cyclin A. Phosphopeptide mapping indicated that Cdk2-Speedy/Ringo A2 and Cdk2-cyclin A phosphorylate distinct subsets of sites on Cdc25 proteins. Thus, the low activity that Cdk2-Speedy/Ringo A2 displays when assayed on conventional Cdk2 substrates may significantly underestimate the potential physiological importance of Cdk2-Speedy/Ringo A2 in phosphorylating key subsets of Cdk2 substrates. Unlike Cdk2-cyclin A, whose activity depends strongly on activating phosphorylation of Cdk2 on Thr-160, neither the overall catalytic activity nor the substrate recognition by Cdk2-Speedy/Ringo A2 was significantly affected by this phosphorylation. Furthermore, Cdk2-Speedy/Ringo A2 was not a suitable substrate for metazoan CAK (which phosphorylates Cdk2 at Thr-160), supporting the notion that Speedy/Ringo A2 activates Cdk2 in a CAK-independent manner. There are major differences in substrate preferences between CDK-Speedy/Ringo A2 and Cdk2-cyclin complexes. These differences may accommodate the CAK-independent activation of Cdk2 by Speedy/Ringo A2 and they raise the possibility that CDK-Speedy/Ringo A2 complexes could phosphorylate and regulate a subset of non-canonical CDK substrates, such as Cdc25 protein phosphatases, to control cell cycle progression.
Figure 1. Characterization of [unP]Cdk2, [pT160]Cdk2, Speedy/Ringo A2, and K2A2coexp. (A) Activation of [unP]Cdk2 and [pT160]Cdk2 by cyclin A. 0.05 μg of [unP]Cdk2 or [pT160]Cdk2 was preincubated with the indicated amounts of cyclin A prior to determination of its histone H1 kinase activity. (B) Activation of [unP]Cdk2 and [pT160]Cdk2 by Speedy/Ringo A2. 0.5 μg of [unP]Cdk2 or [pT160]Cdk2 was preincubated with the indicated amounts of GST-Speedy/Ringo A2 prior to determination of its histone H1 kinase activity. (C) Gel filtration analysis of K2A2coexp. Cdk2 and Speedy/Ringo A2-His6 were coexpressed in E. coli and purified on a metal affinity column. The purified K2A2coexp was loaded on a Superdex-200 column; one-ml fractions were collected. Proteins from 20 μl of the input (lane L) or from 40 μl of fractions 9–21 were resolved by SDS-PAGE, transferred to a PVDF membrane, and detected with a Cdk2-specific antibody (lower panel), or by staining with Coomassie Brilliant Blue R-250 (upper panel). (D) Time course of ATPase activity of [pT160]Cdk2-cyclin A and K2A2coexp. 16.7 μM of [pT160]Cdk2-cyclin A or K2A2coexp was incubated with [γ-32P]ATP for the indicated times. Samples were chromatographed to resolve 32Pi from [γ-32P]ATP. The rate of ATP hydrolysis was quantitated by phosphorimaging analysis. (E) The relative ATPase activities of [pT160]Cdk2-cyclin A and K2A2coexp were calculated from the slopes in panel D. The ATPase activity of [pT160]Cdk2-cyclin A was set to 100%.
Figure 2. Effects of alanine substitutions at each of the three charged positions in a KSPRK substrate on relative phosphorylation by Cdk2-cyclin A and Cdk2-Speedy/Ringo A2. The phosphorylation efficiencies of the indicated GST peptides by K2A2coexp, [unP]Cdk2-Speedy/Ringo A2, [pT160]Cdk2-Speedy/Ringo A2, and [pT160]Cdk2-cyclin A were compared. Assays were performed at substrate concentrations of 50 μM. All values are relative to the phosphorylation of the wild type (KSPRK) substrate by the same enzyme. Values represent the means ± S.E. from three separate experiments.
Figure 3. Effects of amino acid substitutions at the +3 position of KSPRK on substrate utilization by [unP]Cdk2-Speedy/Ringo A2, [unP]Cdk2-cyclin A, K2A2coexp, and [pT160]Cdk2-cyclin A. (A) Comparison of the substrate specificity of [unP]Cdk2-Speedy/Ringo A2 (open bars) and [unP]Cdk2-cyclin A (solid bars). Assays were performed at substrate concentrations of 50 μM. All phosphorylation efficiencies are relative to the phosphorylation of the KSPRK substrate by the same enzyme. Values represent the means ± S.E. from three separate experiments. (B) Comparison of the substrate specificities of [unP]Cdk2-Speedy/Ringo A2, K2A2coexp, and [pT160]Cdk2-Speedy/Ringo A2. Assays were performed at substrate concentrations of 50 μM. All phosphorylation efficiencies are relative to the phosphorylation of the KSPRK substrate by the same enzyme. Values represent the means ± S.E. from three separate experiments. Single letters indicate the amino acid at the +3 position of KSPRK. H1, histone H1.
Figure 4. Velocity versus concentration plots for phosphorylation of substrates by [unP]Cdk2-Speedy/Ringo A2 and [pT160]Cdk2-Speedy/Ringo A2. Velocity versus concentration plots for [unP]Cdk2-Speedy/Ringo A2 (A) and [pT160]Cdk2-Speedy/Ringo A2 (B). The substrates are histone H1 (solid circles), KSPRK (solid squares), KSPRR (open squares), and KSPRY (open circles).
Figure 5. Phosphorylation of Cdc25A, B, and C by Cdk2-Speedy/Ringo A2. (A) Potential CDK phosphorylation sites in human Cdc25A, B, and C. There are 12 (S/T)PXX sequences in Cdc25A, 14 in Cdc25B, and 6 in Cdc25C. The two sequences that come closest to fitting the consensus CDK phosphorylation sequence (S/T)PX(K/R) are underlined. (B) Phosphorylation of Cdc25A, B, and C by K2A2coexp and [pT160]Cdk2-cyclin A. Phosphorylation of GST-Cdc25A, B, and C (5 μg) by the indicated amounts of K2A2coexp (lanes 1–6) or Cdk2-cyclin A (lanes 7–12). Phosphorylated proteins were separated by SDS-PAGE and detected by autoradiography. (C) Phosphorylation of histone H1 by K2A2coexp and [pT160]Cdk2-cyclin A. Histone H1 (5 μM) was phosphorylated by the indicated amounts of K2A2coexp (lanes 1–6) or Cdk2-cyclin A (lanes 7–12). Phosphorylated proteins were separated by SDS-PAGE and detected by autoradiography.
Figure 6. Trypic phosphopeptide mapping of Cdc25 proteins. Tryptic phosphopeptide mapping of Cdc25 phosphorylated by [pT160]Cdk2-cyclin A (A) and by K2A2coexp (B). GST-Cdc25A, B, and C (5 μg) were phosphorylated by K2A2coexp and [pT160]Cdk2-cyclin A, separated by 10% SDS-PAGE, extracted, and digested with trypsin. Phosphopeptides were separated on thin layer chromatography plates by electrophoresis followed by chromatography and were detected by autoradiography.
Figure 7. Effects of Speedy/Ringo A2 binding on the phosphorylation and dephosphorylation of Cdk2 on Thr-160. (A) Speedy/Ringo A2 hinders the phosphorylation of Cdk2 on Thr-160 by budding yeast Cak1p. GST-Cdk2D145N (1 μg) was incubated with increasing amounts (0, 1, 2.5, 5, and 10 μg) of GST-Speedy/Ringo A2 or GST prior to phosphorylation by Cak1p (100 ng). The reactions were terminated and phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. (B) Speedy/Ringo A2 binding did not stimulate Thr-160 phosphorylation by mammalian CAK. GST-Cdk2D145N (1 μg; lanes 1, 4, 5) was preincubated with cyclin A (1 μg; lanes 2 and 4) or GST-Speedy/Ringo A2 (4 μg; lanes 3 and 5) before phosphorylation by CAK. Samples were processed as described in Methods. (C) Speedy/Ringo A2 hinders the dephosphorylation of Cdk2 on Thr-160 by PP2Cα. [32P-T160]GST-Cdk2D145N was preincubated with cyclin A (lanes 2 and 4), GST-Speedy/Ringo A2 (lanes 6 and 8), or buffer alone (lanes 1, 3, 5, 7) prior to addition of recombinant human PP2Cα (lanes 3, 4, 7, 8) or buffer (lanes 1, 2, 5, 6). [32P-T160]GST-Cdk2D145N was detected by autoradiography.
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