Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.     

An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase.

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.     

RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner

Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.     

Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.     

p53 represses autophagy in a cell cycle-dependent fashion

Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic pifithrin-?), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-XL with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G1 phase, less so in the S phase and spares the G2/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.     

Drosophila SUUR protein associates with PCNA and binds chromatin in a cell cycle-dependent manner

Drosophila SUUR (Suppressor of UnderReplication) protein was shown to regulate the DNA replication elongation process in endocycling cells. This protein is also known to be the component of silent chromatin in polyploid and diploid cells. To mark the different cell cycle stages, we used immunostaining patterns of PCNA, the main structural component of replication fork. We demonstrate that SUUR chromatin binding is dynamic throughout the endocyle in Drosophila salivary glands. We observed that SUUR chromosomal localization changed along with PCNA pattern and these proteins largely co-localized during the late S-phase in salivary glands. The hypothesized interaction between SUUR and PCNA was confirmed by co-immunoprecipitation from embryonic nuclear extracts. Our findings support the idea that the effect of SUUR on replication elongation depends on the cell cycle stage and can be mediated through its physical interaction with replication fork.     

Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6’s interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.

Plant casein kinases coordinate cell cycle by regulating the stability of a cyclin-dependent kinase inhibitor through promoting interaction with E3 ubiquitin ligases and proteasomal degradation by phosphorylation.     

Importin alpha/beta-mediated nuclear protein import is regulated in a cell cycle-dependent manner

Functional nuclear proteins are selectively imported into the nucleus by transport factors such as importins alpha and beta. The relationship between the efficiency of nuclear protein import and the cell cycle was measured using specific import substrates for the importin alpha/beta-mediated pathway. After the microinjection of SV40 T antigen nuclear localization signal (NLS)-containing substrates into the cytoplasm of synchronized culture cells at a certain phase of the cell cycle, the nuclear import of the substrates was measured kinetically. Cell cycle-dependent change in import efficiency, but not capacity, was found. That is, import efficiency was found low in the early S, G2/M, and M/G1 phases compared with other phases. In addition, we found that the extent of co-imunoprecipitation of importin alpha with importin beta from cell extracts was strongly associated with import efficiency. These results indicate that the importin alpha/beta-mediated nuclear import machinery is regulated in a cell cycle-dependent manner through the modulation of interaction modes between importins alpha and beta.     

The plant homologue of MAP kinase is expressed in a cell cycle-dependent and organ-specific manner

In animals, MAP kinase plays a key role in growth factor-stimulated signalling and in mitosis. The isolation of a Medicago sativa cDNA clone MsK7 which shows 52% identity to animal MAP kinases is reported. The deduced protein sequence shows all the important structural features of MAP kinases and also contains the highly conserved Thr-183 and Tyr-185 residues. Northern analysis of synchronized alfalfa cells showed that the MsK7 kinase gene is expressed at low levels in G1 phase but at higher levels in S and G2 phases of the cell cycle. In the plant, only stems and roots were found to contain MAP kinase MsK7 mRNA. Southern and PCR analyses indicated that alfalfa contains at least four highly related MAP kinase genes.     

Mycobacterium tuberculosis serine/threonine kinases PknB, PknD, PknE, and PknF phosphorylate multiple FHA domains

The physiologic roles and the substrates of the Mycobacterium tuberculosis (Mtb) serine/threonine kinases are largely unknown. Here, we report six novel interactions of PknB, PknD, PknE, and PknF with the Forkhead-Associated (FHA) domains of Rv0020c and the putative ABC transporter Rv1747. Purified PknB and PknF kinase domains phosphorylated multiple FHA-domain proteins in vitro. Although they remain to be verified in vivo, these reactions suggest a web of interactions between STPKs and FHA domains.     

The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis

The WAG1 and WAG2 genes of Arabidopsis thaliana encode protein-serine/threonine kinases that are closely related to PINOID. In order to determine what roles WAG1 and WAG2 play in seedling development, we used a reverse genetics approach to study the wag1, wag2 and wag1/wag2 mutant phenotypes for clues. Although the wag mutants do not contain detectable amounts of the corresponding mRNA, they are wild type in most respects. However, wag1/wag2 double mutants exhibit a pronounced wavy root phenotype when grown vertically on agar plates, a phenotype observed in wild-type plants only on plates inclined to angles less than 90 degrees. The wag1 and wag2 mutants also demonstrate enhanced root waving, but to a lesser extent. Moreover, the double mutant roots are more resistant to the effects of N-1-naphthylphthalamic acid on the inhibition of root curling, raising the possibility that transport of auxin is affected in the wag mutants. Promoter fusions to the gusA reporter gene demonstrate that the WAG promoters are most active in root tips, consistent with the observed phenotypes in the wag mutants.     

Insulin-activated protein kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the p70 S6 kinase

p70 S6 kinase, a major insulin-mitogen-activated ribosomal S6 protein kinase in mammalian cells, is activated by phosphorylation of multiple Ser/Thr residues on the enzyme polypeptide. A synthetic peptide, corresponding to a 37-residue segment from the carboxyl-terminal tail of the kinase which resembles the sequence phosphorylated in S6, acts as a competitive inhibitor of p70 S6 kinase without itself being phosphorylated by the enzyme. This synthetic peptide is phosphorylated by an array of protein kinases which are rapidly activated by insulin. Thus, these sequences of p70 S6 kinase constitute a potential autoinhibitory pseudosubstrate site, whose phosphorylation is catalyzed by candidate upstream-activating protein kinases.     

Src and FAK kinases cooperate to phosphorylate paxillin kinase linker, stimulate its focal adhesion localization, and regulate cell spreading and protrusiveness

The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility.