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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 9â21 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 1â6) or Cdk2-cyclin A (lanes 7â12). 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 1â6) or Cdk2-cyclin A (lanes 7â12). 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.
Adams,
Retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin-cdk complexes.
1999, Pubmed
Adams,
Retinoblastoma protein contains a C-terminal motif that targets it for phosphorylation by cyclin-cdk complexes.
1999,
Pubmed
Archambault,
Two-faced cyclins with eyes on the targets.
2005,
Pubmed
Brown,
Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.
1999,
Pubmed
Brown,
The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases.
1999,
Pubmed
Busino,
Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis.
2004,
Pubmed
Cans,
Proteasome-dependent degradation of human CDC25B phosphatase.
1999,
Pubmed
Chen,
Cyclin-binding motifs are essential for the function of p21CIP1.
1996,
Pubmed
,
Xenbase
Cheng,
Identification and comparative analysis of multiple mammalian Speedy/Ringo proteins.
2005,
Pubmed
,
Xenbase
Cheng,
Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms.
2000,
Pubmed
Cheng,
Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases.
1999,
Pubmed
El-Guindy,
Phosphorylation of Epstein-Barr virus ZEBRA protein at its casein kinase 2 sites mediates its ability to repress activation of a viral lytic cycle late gene by Rta.
2004,
Pubmed
Espinoza,
A cyclin-dependent kinase-activating kinase (CAK) in budding yeast unrelated to vertebrate CAK.
1996,
Pubmed
Ferby,
A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in Xenopus oocytes.
1999,
Pubmed
,
Xenbase
Fesquet,
The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues.
1993,
Pubmed
,
Xenbase
Hagopian,
Kinetic basis for activation of CDK2/cyclin A by phosphorylation.
2001,
Pubmed
Holmes,
A predictive scale for evaluating cyclin-dependent kinase substrates. A comparison of p34cdc2 and p33cdk2.
1996,
Pubmed
,
Xenbase
Holmes,
The role of Thr160 phosphorylation of Cdk2 in substrate recognition.
2001,
Pubmed
Kaldis,
The Cdk-activating kinase (CAK) from budding yeast.
1996,
Pubmed
Kaldis,
Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities.
1998,
Pubmed
Kaldis,
The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine.
2000,
Pubmed
Karaiskou,
Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.
2001,
Pubmed
,
Xenbase
King,
How proteolysis drives the cell cycle.
1996,
Pubmed
Lenormand,
Speedy: a novel cell cycle regulator of the G2/M transition.
1999,
Pubmed
,
Xenbase
Levine,
Saccharomyces cerevisiae G1 cyclins differ in their intrinsic functional specificities.
1996,
Pubmed
Loog,
Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates.
2005,
Pubmed
Morgan,
The dynamics of cyclin dependent kinase structure.
1996,
Pubmed
Morgan,
Cyclin-dependent kinases: engines, clocks, and microprocessors.
1997,
Pubmed
Nigg,
The substrates of the cdc2 kinase.
1991,
Pubmed
Nilsson,
Cell cycle regulation by the Cdc25 phosphatase family.
2000,
Pubmed
Peeper,
A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes.
1993,
Pubmed
Pines,
Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport.
1991,
Pubmed
Pines,
Cyclins and cyclin-dependent kinases: a biochemical view.
1995,
Pubmed
Poon,
The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.
1993,
Pubmed
,
Xenbase
Porter,
Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of Cdk2.
2002,
Pubmed
,
Xenbase
Saha,
p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases.
1997,
Pubmed
,
Xenbase
Schulman,
Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A.
1998,
Pubmed
Sherr,
Inhibitors of mammalian G1 cyclin-dependent kinases.
1995,
Pubmed
Sherr,
CDK inhibitors: positive and negative regulators of G1-phase progression.
1999,
Pubmed
Solomon,
Regulation of CDKs by phosphorylation.
1998,
Pubmed
Solomon,
Role of phosphorylation in p34cdc2 activation: identification of an activating kinase.
1992,
Pubmed
,
Xenbase
Solomon,
Cyclin activation of p34cdc2.
1990,
Pubmed
,
Xenbase
Solomon,
CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15.
1993,
Pubmed
,
Xenbase
Solomon,
Activation of the various cyclin/cdc2 protein kinases.
1993,
Pubmed
Song,
Phosphoprotein-protein interactions revealed by the crystal structure of kinase-associated phosphatase in complex with phosphoCDK2.
2001,
Pubmed
Songyang,
Use of an oriented peptide library to determine the optimal substrates of protein kinases.
1994,
Pubmed
Stevenson,
Activation mechanism of CDK2: role of cyclin binding versus phosphorylation.
2002,
Pubmed
Tarricone,
Structure and regulation of the CDK5-p25(nck5a) complex.
2001,
Pubmed
Thuret,
Civ1 (CAK in vivo), a novel Cdk-activating kinase.
1996,
Pubmed
Tyers,
Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins.
1993,
Pubmed
Zhu,
The pRB-related protein p107 contains two growth suppression domains: independent interactions with E2F and cyclin/cdk complexes.
1995,
Pubmed
