The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.     

Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition

The G2-M transition of the cell cycle is triggered by the p34(cdc2)/cyclin B kinase. During the prophase/metaphase transition, the inactive, Thr-14/Tyr-15 phosphorylated form of p34(cdc2) (TP-YP) is modified to an active, Thr-14/Tyr-15 dephosphorylated form (T-Y) by the cdc25 dual-specificity phosphatase. Using highly synchronized starfish oocytes as a cellular model, we show that dephosphorylation in vivo and in vitro occurs in two steps: Thr-14 dephosphorylation precedes Tyr-15 dephosphorylation. The transient intermediate form (T-YP), which can be obtained in vitro by treatment of TP-YP by protein phosphatase 2A, displays low but significant kinase activity. These results raise the possibility that the intermediate form T-YP may be involved in the autocatalytic amplification of the p34(cdc2)/cyclin B complex through phosphorylation/activation of the cdc25 phosphatase and phosphorylation/inactivation of the wee1 kinase.     

Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2

Subcellular localization of type II cAMP-dependent protein kinase is determined by the interactions of the regulatory subunit (RII) with specific RII-anchoring proteins. By using truncated NH2-terminal RII beta fusion proteins expressed in Escherichia coli and the mitotic protein kinase p34cdc2 isolated from HeLa cells or starfish oocytes, we investigated the in vitro phosphorylation of RII beta by these kinases. The putative site for phosphorylation by the mitotic kinases is Thr-69 in the NH2-terminal domain of RII beta. This phosphorylation site matches the consensus sequence X(T/S)PX(K/R) for p34cdc2 recognition and belongs to a well-conserved sequence found in all RII beta sequences known to date. In contrast to phosphorylation by casein kinase II or the cAMP-dependent protein kinase catalytic subunit, phosphorylation of RII beta by mitotic kinases impaired its interaction with a well-known RII-anchoring protein, the neuronal microtubule-associated protein 2. The potential regulatory significance of the phosphorylation of this site on the interaction with microtubule-associated protein 2 and other RII-anchoring proteins and the physiological relevance of this cyclin B/p34cdc2 kinase-catalyzed modification of RII beta (or phosphorylation by other proline-directed protein kinases) are discussed.     

cdc2-like kinase from rat spinal cord specifically phosphorylates KSPXK motifs in neurofilament proteins: isolation and characterization

A protein kinase that phosphorylates a specific KSP sequence [K(S/T)PXK], which is abundant in high molecular weight neurofilament (NF) proteins, was identified and isolated from rat spinal cord. Characterization of this enzyme activity revealed a close relationship with p34cdc2 kinase with respect to its molecular mass (32.5 kDa by SDS/PAGE) and substrate specificities. It could phosphorylate a synthetic peptide analog of the simian virus 40 large tumor antigen, reportedly a specific substrate for p34cdc2 kinase. Histone (H1) and peptide analogs of the KSP sequence present in the C-terminal end of rat and mouse neurofilament proteins were phosphorylated. This kinase did not phosphorylate alpha-casein and peptide substrates of other known second messenger-dependent or -independent kinases. Dephosphorylated rat NF protein NF-H was strongly phosphorylated by the purified enzyme; NF proteins NF-M and native NF-H, but not NF-L, were slightly phosphorylated. Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate. On Western blots, the enzyme was immunoreactive with antibody against the C-terminal end of cdc2 kinase (mouse) and neuronal cdc2-like kinase from rat but not with an antibody against the conserved PSTAIRE region of the p34cdc2 kinase. The antibody against the C-terminal end of cdc2 kinase could immunoprecipitate (immunodeplete) the purified kinase activity. Since the adult nervous system is composed primarily of postmitotic cells, the present observations indicate a nonmitotic role for this cdc2-like kinase activity. The effective phosphorylation of NF-H by this kinase suggests a function in axonal structure.     

Ribonucleotide reductase R2 protein is phosphorylated at serine-20 by P34cdc2 kinase

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.     

Effect of suramin on p34cdc2 kinase in vitro and in extracts from human H69 cells: evidence for a double mechanism of action

We examined the effect of suramin, an anticancer agent and a functional analog of naturally occuring glycosaminoglycans, on p34cdc2 kinase. We find that suramin strongly inhibits the catalytic activity of purified p34cdc2 kinase (IC50 approximately 4 microM), whereas it only weakly inhibits the p13-agarose precipitated kinase activity from nuclear and cytoplasmic extracts of the asynchronous H69 human small cell lung cancer cells. We also find that the tyrosine phosphorylation of p34cdc2 kinase in the nuclear extract is increased about twice when the extracts are preincubated with 50 microM of suramin prior to the p13-agarose precipitation. We propose that this increase might result from the inhibitory effect of suramin towards p34cdc2-specific tyrosine phosphatases. These results suggest both a direct and an indirect effect of suramin on p34cdc2 kinase. We also find that heparin is a potent inhibitor of purified cdc2 kinase (IC50 approximately 3.5 micrograms/ml). Therefore, glycosaminoglycans might be physiological regulators of p34cdc2 kinase in vivo.     

Regulation of V(D)J recombination activator protein RAG-2 by phosphorylation

Antigen receptor genes are assembled by site-specific DNA rearrangement. The recombination activator genes RAG-1 and RAG-2 are essential for this process, termed V(D)J rearrangement. The activity and stability of the RAG-2 protein have now been shown to be regulated by phosphorylation. In fibroblasts RAG-2 was phosphorylated predominantly at two serine residues, one of which affected RAG-2 activity in vivo. The threonine at residue 490 was phosphorylated by p34cdc2 kinase in vitro; phosphorylation at this site in vivo was associated with rapid degradation of RAG-2. Instability was transferred to chimeric proteins by a 90-residue portion of RAG-2. Mutation of the p34cdc2 phosphorylation site of the tumor suppressor protein p53 conferred a similar phenotype, suggesting that this association between phosphorylation and degradation is a general mechanism.     

Fission yeast Csk1 is a CAK-activating kinase (CAKAK)

Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK).     

Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase Plx1

The Xenopus polo-like kinase 1 (Plx1) is essential during mitosis for the activation of Cdc25C, for spindle assembly, and for cyclin B degradation. Polo-like kinases from various organisms are activated by phosphorylation by an unidentified protein kinase. A protein kinase, polo-like kinase kinase 1 or xPlkk1, that phosphorylates and activates Plx1 in vitro was purified to near homogeneity and cloned. Phosphopeptide mapping of Plx1 phosphorylated in vitro by recombinant xPlkk1 or in progesterone-treated oocytes indicates that xPlkk1 may activate Plx1 in vivo. The xPlkk1 protein itself was also activated by phosphorylation on serine and threonine residues, and the kinetics of activation of xPlkk1 in vivo closely paralleled the activation of Plx1. Moreover, microinjection of xPlkk1 into Xenopus oocytes accelerated the timing of activation of Plx1 and the transition from G2 to M phase of the cell cycle. These results define a protein kinase cascade that regulates several events of mitosis.     

G1 cyclin-dependent activation of p34CDC28 (Cdc28p) in vitro

In Saccharomyces cerevisiae, transient accumulation of G1 cyclin/p34CDC28 (Cdc28p) complexes induces cells to traverse the cell cycle Start checkpoint and commit to a round of cell division. To investigate posttranslational controls that modulate Cdc28p activity during the G1 phase, we have reconstituted cyclin-dependent activation of Cdc28p in a cyclin-depleted G1 extract. A glutathione S-transferase-G1 cyclin chimera (GST-Cln2p) efficiently binds to and activates Cdc28p as a histone H1 kinase. Activation of Cdc28p by GST-Cln2p requires ATP, crude yeast cytosol, and the conserved Thr-169 residue that serves in other organisms as a substrate for phosphorylation by cyclin-dependent protein kinase-activating kinase. This assay may be useful for distinguishing genes that promote directly the posttranslational assembly of active Cln2p/Cdc28p kinase complexes from those that stimulate the accumulation of active complexes via a positive-feedback loop that governs synthesis of G1 cyclins.     

Cloning and characterization of RLPK, a novel RSK-related protein kinase

A novel protein kinase whose activity can be stimulated by mitogen in vivo was cloned and characterized. The cDNA of this gene encodes an 802-amino acid protein (termed RLPK) with the highest homology (37% identity) to the two protein kinase families, p90(RSK) and p70(RSK). Like p90(RSR), but not p70(RSK), RLPK also contains two complete nonidentical protein kinase domains. RLPK mRNA is widely expressed in all human tissues examined and is enriched in the brain, heart, and placenta. In HeLa cells, transiently expressed epitope-tagged RLPK can be strongly induced by epidermal growth factor, serum, and phorbol 12-myristate 13-acetate, but only moderately up-regulated by tumor necrosis factor-alpha and other stress-related stimuli. The activity of RLPK stimulated by epidermal growth factor was not inhibited by several known protein kinase C inhibitors nor by rapamycin, a known specific inhibitor for p70(RSK), but could be inhibited by herbimycin A, a tyrosine kinase inhibitor, and partially inhibited by PD98059 or SB203580, inhibitors for the mitogen-activated protein kinase pathways. Recombinant RLPK possesses high phosphorylation activity toward histone 2B and the S6 peptide, RRRLSSLRA. Although purified recombinant RLPK can be phosphorylated by ERK2 and p38alpha in vitro, its activity is not affected by this phosphorylation. Moreover, the treatment of RLPK with acid phosphatase did not reduce its in vitro kinase activity. These data suggest that RLPK is structurally similar to previously isolated RSKs, but its regulatory mechanism may be distinct from either p70(RSK) or p90(RSK)s.     

MAP kinase links the fertilization signal transduction pathway to the G1/S-phase transition in starfish eggs

The mechanism by which fertilization initiates S-phase in the zygote is examined by manipulating the activity of MAP kinase in mature starfish eggs. These unfertilized eggs, which are arrested at G1-phase after the completion of meiosis, have high MAP kinase activity but undetectable cdc2 kinase activity. Either fertilization or inhibition of protein synthesis causes a decrease in MAP kinase activity, which is followed by DNA synthesis. Inactivation of MAP kinase with its specific phosphatase, CL100, initiates DNA synthesis in the absence of fertilization, while constitutive activation of MAP kinase with MEK represses the initiation of DNA synthesis following fertilization. Thus, in unfertilized mature starfish eggs, a capacity for DNA replication is already acquired, but entry into S-phase is negatively regulated by MAP kinase activity that is supported by a continuously synthesized protein(s) but not by cdc2 kinase. Upon fertilization, downregulation of MAP kinase activity is necessary and sufficient for triggering the G1/S-phase transition.     

Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of Acanthamoeba myosin I heavy chain kinase

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower kcat and higher Km for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser/Thr protein kinases as well as for the activity of MIHCK.     

Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.     

Control of cell division in eucaryotes

In eucaryotes, M-phase promoting factor (MPF) triggers meiosis in germ cells and mitosis in somatic cells. MPF is composed of two proteins of which one is homologous with the protein kinase encoded by gene cdc2 of Schizosaccharomyces pombe (p34cdc2) and the other is a cyclin whose concentration oscillates during the cell cycle. Inactivation of p34cdc2 (MPF) requires cyclin degradation, which occurs during the metaphase-anaphase transition of the M-phase. Cyclin degradation is not only associated with cell cycle progression, but is also required for this event. At the G2/M transition, p34cdc2 protein kinase is activated and catalyzes phosphorylation of numerous key proteins, thus enabling cell changes to occur. p34cdc2 undergoes multiple-site phosphorylation in a cell cycle-dependent manner. At onset of mitosis, the protein phosphatase cdc25 catalyzes dephosphorylation of the p34cdc2 kinase at the threonine 14 and tyrosine 15 sites. This event may be the rate-limiting step controlling onset of mitosis in cells of vertebrates. A second protein kinase, encoded by the proto-oncogene c-mos, acts as a cytostatic factor preventing cyclin degradation and keeping unfertilized eggs from progressing beyond the second meiotic metaphase.     

Inhibition of small G proteins by clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes

The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD.     

Detection of designer drugs in human hair by ion mobility spectrometry (IMS)

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP-dependent kinase creates the more acidic species.