The Xenopus Suc1/Cks Protein Promotes the Phosphorylation of G2/M Regulators

The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF). In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1. At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself. The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown. We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex. Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts. We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B. This observation could explain why overexpression of Pin1 inhibits mitotic initiation. These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2

Cell Cycle Control by Xenopus p28^(Kix1) a Developmentally Regulated Inhibitor of Cyclin-dependent Kinases

We have isolated Xenopus p28^(Kix1), a member of the p21^(CIP1)/p27^(KIP1)/p57^(KIP2) family of cyclin-dependent kinase (Cdk) inhibitors. Members of this family negatively regulate cell cycle progression in mammalian cells by inhibiting the activities of Cdks. p28 shows significant sequence homology with p21, p27, and p57 in its N-terminal region, where the Cdk inhibition domain is known to reside. In contrast, the C-terminal domain of p28 is distinct from that of p21, p27, and p57. In co-immunoprecipitation experiments, p28 was found to be associated with Cdk2, cyclin E, and cyclin A, but not the Cdc2/cyclin B complex in Xenopus egg extracts. Xenopus p28 associates with the proliferating cell nuclear antigen, but with a substantially lower affinity than human p21. In kinase assays with recombinant Cdks, p28 inhibits pre-activated Cdk2/cyclin E and Cdk2/cyclin A, but not Cdc2/cyclin B. However, at high concentrations, p28 does prevent the activation of Cdc2/cyclin B by the Cdk-activating kinase. Consistent with the role of p28 as a Cdk inhibitor, recombinant p28 elicits an inhibition of both DNA replication and mitosis upon addition to egg extracts, indicating that it can regulate multiple cell cycle transitions. The level of p28 protein shows a dramatic developmental profile: it is low in Xenopus oocytes, eggs, and embryos up to stage 11, but increases approximately ~ 100-fold between stages 12 and 13, and remains high thereafter. The induction of p28 expression temporally coincides with late gastrulation. Thus, although p28 may play only a limited role during the early embryonic cleavages, it may function later in development to establish a somatic type of cell cycle. Taken together, our results indicate that Xenopus p28 is a new member of the p21/p27/p57 class of Cdk inhibitors, and that it may play a role in developmental processes

Cell cycle regulation of a Xenopus Wee1-like kinase

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr- 15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase

The Xenopus Chk1 Protein Kinase Mediates a Caffeine-sensitive Pathway of Checkpoint Control in Cell-free Extracts

We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action of a distinct Ser-287-specific kinase in addition to Xchk1. Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1

Current Address: Queensland Institute of Medical Research, Ban-croft Centre, 300 Herson Rd

Human Cdc25C is a protein phosphatase that dephosphorylates and activates Cdc2-cyclin B to trigger entry into mitosis. Cdc25C is itself regulated by phosphorylation. In asynchronously growing HeLa cells, we have determined that serine 216 is the major site of Cdc25C phosphorylation. We have isolated a protein kinase that binds to Cdc25C and phosphorylates serine 216. "he kinase binds within amino acids 20&256 of Cdc25C. This region is conserved in some Cdc25 homologues and contains a putative bipartite nuclear localization signal just downstream from serine 216. Finally, the Cdc25C-associating kinase was purified over 8000-fold from rat liver as a 36-38-kDa doublet of proteins. Progression through the eukaryotic cell cycle involves the sequential activation of cyclin-dependent kinases (Cdks)' (reviewed in Refs. 1 and 2). As the name implies, activation of a Cdk is dependent upon its association with a cyclin regulatory subunit. Cdk-cyclin complexes can also be regulated by reversible phosphorylation, which helps to ensure the proper timing of activation during the cell cycle (reviewed in Ref. 3). Entry of cells into mitosis is regulated in part by the activity of the Cdc2-cyclin B complex, which serves as a paradigm for the Cdk family. Cyclin B is synthesized and associates with Cdc2 in the cytoplasm beginning in S-phase and continuing throughout G,-phase (4). In higher eukaryotic cells, cyclin B association induces the rapid phosphorylation of Cdc2 on three sites. Phosphorylation on threonine 161 is required for kinase activity, whereas phosphorylation on threonine 14 and tyrosine 15 suppresses kinase activity (5-12). I t is the phosphorylation of threonine 14 and tyrosine 15 that maintains Cdc2-cyclin B in an inactive state. The kinase responsible for phosphorylating threonine 14 has not been identified, but two kinases, Weel and Mikl, have been identified that regulate the phosphorylation of tyrosine 15 (7-9, 13-15). Dephosphorylation of threonine 14 and tyrosine 15 is the final step in the activation of Cdc2-cyclin B and is required for entry of cells into mitosis (6, 314-362-7463. Dephosphorylation and activation of Cdc2-cyclin B is dependent upon the Cdc25 phosphatase. Cdc25 was first identified as a cell cycle mutant in Schizosaccharomyces pombe that acts antagonistically to Weel The Cdc25C protein is present throughout the cell cycle in higher eukaryotes, whereas its substrate (Cdc2-cyclin B) accumulates throughout the S-and G,-phases of the cell cycle Phosphorylation has been found to directly activate the enzymatic activity of Cdc25C 3046

Skeletal muscle cells lacking the retinoblastoma protein display defects in muscle gene expression and accumulate in S and G2 phases of the cell cycle.

Viral oncoproteins that inactivate the retinoblastoma tumor suppressor protein (pRb) family both block skeletal muscle differentiation and promote cell cycle progression. To clarify the dependence of terminal differentiation on the presence of the different pRb-related proteins, we have studied myogenesis using isogenic primary fibroblasts derived from mouse embryos individually deficient for pRb, p107, or p130. When ectopically expressed in fibroblasts lacking pRb, MyoD induces an aberrant skeletal muscle differentiation program characterized by normal expression of early differentiation markers such as myogenin and p21, but attenuated expression of late differentiation markers such as myosin heavy chain (MHC). Similar defects in MHC expression were not observed in cells lacking either p107 or p130, indicating that the defect is specific to the loss of pRb. In contrast to wild-type, p107-deficient, or p130-deficient differentiated myocytes that are permanently withdrawn from the cell cycle, differentiated myocytes lacking pRb accumulate in S and G2 phases and express extremely high levels of cyclins A and B, cyclin-dependent kinase (Cdk2), and Cdc2, but fail to readily proceed to mitosis. Administration of caffeine, an agent that removes inhibitory phosphorylations on inactive Cdc2/cyclin B complexes, specifically induced mitotic catastrophe in pRb-deficient myocytes, consistent with the observation that the majority of pRb-deficient myocytes arrest in S and G2. Together, these findings indicate that pRb is required for the expression of late skeletal muscle differentiation markers and for the inhibition of DNA synthesis, but that a pRb-independent mechanism restricts entry of differentiated myocytes into mitosis

Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A.

Before fertilization, vertebrate eggs are arrested in meiosis II by cytostatic factor (CSF), which holds the anaphase-promoting complex (APC) in an inactive state. It was recently reported that Mos, an integral component of CSF, acts in part by promoting the Rsk-mediated phosphorylation of the APC inhibitor Emi2/Erp1. We report here that Rsk phosphorylation of Emi2 promotes its interaction with the protein phosphatase PP2A. Emi2 residues adjacent to the Rsk phosphorylation site were important for PP2A binding. An Emi2 mutant that retained Rsk phosphorylation but lacked PP2A binding could not be modulated by Mos. PP2A bound to Emi2 acted on two distinct clusters of sites phosphorylated by Cdc2, one responsible for modulating its stability during CSF arrest and one that controls binding to the APC. These findings provide a molecular mechanism for Mos action in promoting CSF arrest and also define an unusual mechanism, whereby protein phosphorylation recruits a phosphatase for dephosphorylation of distinct sites phosphorylated by another kinase

N-(INDAZOLYL)BENZAMIDO DERIVATIVES AS CDK1 INHIBITORS: DESIGN, SYNTHESIS, BIOLOGICAL ACTIVITY, AND MOLECULAR DOCKING STUDIES

A series of N-1H-indazole-1-carboxamides have been synthesized and their effects on both CDK1/cyclin B and K-562 (human chronic myelogenus leukemia) cell line were evaluated. Using a computational model we have observed that all the most active compound 9e,f,i-n exhibited the same binding mode of Purvanalol A in the ATP-binding cleft. Although they were able to moderately inhibit the leukemic cell line K-562, and to show inhibitory activity against the Cdc2-Cyclin B kinase in the low micromolar range, they resulted non cytotoxic against HuDe (IZSL), primary cell cultures from human derm. These preliminary results are quite encouraging in view of the low toxicity showed by the above mentioned compounds

Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells

The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B, Here we present data that the cdc25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, cdc25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when cdc25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of cdc25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic cdc25B activity, and that cdc25C regulates nuclear G(2)/M events, Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events