PDK1 in apical signaling endosomes participates in the rescue of the polarity complex atypical PKC by intermediate filaments in intestinal epithelia

Phosphorylation of the activation domain of protein kinase C (PKC) isoforms is essential to start a conformational change that results in an active catalytic domain. This activation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This "rescue" mechanism is responsible for the maintenance of the steady-state levels of atypical PKC (aPKC [PKCι/λ and ζ]) and is blocked in inflammation. Although there is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized molecules, it is unclear what kinase performs that function during the rescue and where the rescue takes place. To identify the activating kinase during the rescue mechanism, we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool. PDK1 knockdown and two different PDK1 inhibitors-BX-912 and a specific pseudosubstrate peptide-destabilized PKCι. PDK1 coimmunoprecipitated with PKCι in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays using immunodepletion and rescue with recombinant protein. Surprisingly, we found that in Caco-2 epithelial cells and intestinal crypt enterocytes PDK1 distributes to an apical membrane compartment comprising plasma membrane and apical endosomes, which, in turn, are in close contact with intermediate filaments. PDK1 comigrated with the Rab11 compartment and, to some extent, with the transferrin compartment in sucrose gradients. PDK1, pT555-aPKC, and pAkt were dependent on dynamin activity. These results highlight a novel signaling function of apical endosomes in polarized cells.     

Phosphoinositide-dependent phosphorylation of PDK1 regulates nuclear translocation

3-phosphoinositide-dependent kinase 1 (PDK1) phosphorylates the activation loop of a number of protein serine/threonine kinases of the AGC kinase superfamily, including protein kinase B (PKB; also called Akt), serum and glucocorticoid-induced kinase, protein kinase C isoforms, and the p70 ribosomal S6 kinase. PDK1 contains a carboxyl-terminal pleckstrin homology domain, which targets phosphoinositide lipids at the plasma membrane and is central to the activation of PKB. However, PDK1 subcellular trafficking to other compartments is not well understood. We monitored the posttranslational modifications of PDK1 following insulin-like growth factor 1 stimulation. PDK1 underwent rapid and transient phosphorylation on S396, which was dependent upon plasma membrane localization. Phosphorylation of S396 was necessary for nuclear shuttling of PDK1, possibly through its influence on an adjacent nuclear export sequence. Thus, mitogen-stimulated phosphorylation of PDK1 provides a means for directed PDK1 subcellular trafficking, with potential implications for PDK1 signaling.     

Glycogen synthase kinase-3 couples AKT-dependent signaling to the regulation of p21Cip1 degradation.

Signaling via the phosphoinositide 3-kinase (PI3K)/AKT pathway is crucial for the regulation of endothelial cell (EC) proliferation and survival, which involves the AKT-dependent phosphorylation of the DNA repair protein p21(Cip1) at Thr-145. Because p21(Cip1) is a short-lived protein with a high proteasomal degradation rate, we investigated the regulation of p21(Cip1) protein levels by PI3K/AKT-dependent signaling. The PI3K inhibitors Ly294002 and wortmannin reduced p21(Cip1) protein abundance in human umbilical vein EC. However, mutation of the AKT site Thr-145 into aspartate (T145D) did not increase its protein half-life. We therefore investigated whether a kinase downstream of AKT regulates p21(Cip1) protein levels. In various cell types, AKT phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). Upon serum stimulation of EC, GSK-3beta was phosphorylated at Ser-9. Site-directed mutagenesis revealed that GSK-3 in vitro phosphorylated p21(Cip1) specifically at Thr-57 within the Cdk binding domain. Overexpression of GSK-3beta decreased p21(Cip1) protein levels in EC, whereas the specific inhibition of GSK-3 with lithium chloride interfered with p21(Cip1) degradation and increased p21(Cip1) protein about 10-fold in EC and cardiac myocytes (30 mm, p < 0.001). These data indicate that GSK-3 triggers p21(Cip1) degradation. In contrast, stimulation of AKT increases p21(Cip1) via inhibitory phosphorylation of GSK-3.     

Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (DeltaPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas DeltaPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or DeltaPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3alpha/beta, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither DeltaPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3alpha/beta pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or DeltaPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.     

PKCδ survival signaling in cells containing an activated p21Ras protein requires PDK1

Protein kinase C δ (PKCδ) modulates cell survival and apoptosis in diverse cellular systems. We recently reported that PKCδ functions as a critical anti-apoptotic signal transducer in cells containing activated p21^Ras and results in the activation of AKT, thereby promoting cell survival. How PKCδ is regulated by p21^Ras, however, remains incompletely understood. In this study, we show that PKCδ, as a transducer of anti-apoptotic signals, is activated by phosphotidylinositol 3′ kinase/phosphoinositide-dependent kinase 1 (PI₃K–PDK1) to deliver the survival signal to Akt in the environment of activated p21^Ras. PDK1 is upregulated in cells containing an activated p21Ras. Knock-down of PDK1, PKCδ, or AKT forces cells containing activated p21^Ras to undergo apoptosis. PDK1 regulates PKCδ activity, and constitutive expression of PDK1 increases PKCδ activity in different cell types. Conversely, expression of a kinase-dead (dominant-negative) PDK1 significantly suppresses PKCδ activity. p21^Ras-mediated survival signaling is therefore regulated by via a PI₃K–AKT pathway, which is dependent upon both PDK1 and PKCδ, and PDK1 activates and regulates PKCδ to determine the fate of cells containing a mutated, activated p21^Ras.     

Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase.     

PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).     

Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.     

Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1

3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.     

p21ras mediates control of IL-2 gene promoter function in T cell activation.

It has been shown previously in T cells that stimulation of protein kinase C or the T cell antigen receptor leads to a rapid and persistent activation of p21ras as measured by a dramatic increase in the amount of bound GTP. These stimuli are also known to induce the expression of the T lymphocyte growth factor, interleukin-2 (IL-2), an essential growth factor for the immune system. Receptor induced activation of p21ras has been demonstrated in several cell types but involvement of protein kinase C as an upstream activator of p21ras appears to be unique to T cells. In this study we show that p21ras acts as a component of the protein kinase C and T cell antigen receptor downstream signalling pathway controlling IL-2 gene expression. In the murine T cell line EL4, constitutively active p21ras greatly potentiates the phorbol ester and T cell receptor agonist induced production of IL-2 as measured both by biological assay for the cytokine and by the use of a reporter construct. Active p21ras also partially replaces the requirement for protein kinase C activation in synergizing with a calcium ionophore to induce production of IL-2. Furthermore, using a dominant negative mutant of ras, Ha-rasN17, we show that endogenous ras function is essential for induction of IL-2 expression in response to protein kinase C or T cell receptor stimulation. Activation of ras proteins is thus a necessary but not sufficient event in the induction of IL-2 synthesis. Ras proteins are therefore pivotal signalling molecules in T cell activation.     

cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor.

In fibroblasts, stimulation of receptor tyrosine kinases results in the activation of the extracellular signal-regulated kinase 2 (ERK2). The major signalling pathway employed by these receptors involves the activation of p21ras and raf-1 kinase. Here we show that in NIH3T3 and rat-1 fibroblasts, elevation of the intracellular cAMP level results in the inhibition of ERK2 activation induced by PDGF, EGF and insulin treatment. Analysis of various signalling intermediates shows that cAMP interferes at a site downstream of p21ras, but upstream of raf-1 kinase. Inhibition by cAMP depends on both the cAMP concentration and the absolute amount of p21ras molecules bound to GTP, suggesting a mechanism of competitive inhibition. Also TPA-induced, p21ras-independent, activation of raf-1 kinase and ERK2 is inhibited by cAMP. We have used the inhibitory effect of cAMP to investigate whether phosphorylation of mSos, a p21ras nucleotide exchange factor, is dependent on the activity of the raf-1 kinase/ERK2 pathway. We found that phosphorylation of mSos, as monitored by a mobility shift, is delayed with respect to p21ras and ERK2 activation and is inhibited by cAMP in a similar cell type- and concentration-dependent manner as the inactivation of ERK2. These results provide evidence for a model of p21ras-directed signalling towards ERK2 that feeds back on mSos by regulating its phosphorylation status and that can be negatively modulated by protein kinase A and positively modulated by protein kinase C action.     

Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling.

The mitogen activated protein (MAP) kinase module: (Raf -->MEK-->ERKs) is central to the control of cell growth, cell differentiation and cell survival. The fidelity of signalling and the spatio-temporal activation are key determinants in generating precise biological responses. The fidelity is ensured by scaffold proteins - protein kinase 'insulators' - and by specific docking sites. The duration and the intensity of the response are in part controlled by the compartmentalization of the signalling molecules. Growth factors promote rapid nuclear translocation and persistent activation of p42/p44 MAP kinases, respectively and ERK2/ERK1, during the entire G1 period with an extinction during the S-phase. These features are exquisitely controlled by the temporal induction of the MAP kinase phosphatases, MKP1-3. MKP1 and 2 induction is strictly controlled by the activation of the MAP kinase module providing evidence for an auto-regulatory mechanism. This negative regulatory loop is further enhanced by the capacity of p42/p44 MAPK to phosphorylate MKP1 and 2. This action reduces the degradation rate of MKPs through the ubiquitin-proteasomal system. Whereas the two upstream kinases of the module (Raf and MEK) remain cytoplasmic, ERKs (anchored to MEK in the cytoplasm of resting cells) rapidly translocate to the nucleus upon mitogenic stimulation. This latter process is rapid, reversible and controlled by the strict activation of the MAPK cascade. Following long-term MAPK stimulation, p42/p44 MAPKs progressively accumulate in the nucleus in an inactive form. Therefore we propose that the nucleus represents a site for ERK action, sequestration and signal termination. With the generation of knockdown mice for each of the ERK isoforms, we will illustrate that besides controlling cell proliferation the ERK cascade also controls cell differentiation and cell behaviour.     

PDK1, the master regulator of AGC kinase signal transduction

The interaction of insulin and growth factors with their receptors on the outside surface of a cell, leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) second messenger at the inner surface of the cell membrane. One of the most studied signalling events controlled by PtdIns(3,4,5)P3, comprises the activation of a group of AGC family protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK) and protein kinase C (PKC), which play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Here, we review recent biochemical, genetic and structural studies on the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates and activates the AGC kinase members regulated by PI 3-kinase. We also discuss whether inhibitors of PDK1 might have chemotherapeutic potential in the treatment of cancers in which the PDK1-regulated AGC kinases are constitutively activated.     

Focal adhesion kinase controls cellular levels of p27/Kip1 and p21/Cip1 through Skp2-dependent and -independent mechanisms

Endothelial cell proliferation is a critical step in angiogenesis and requires a coordinated response to soluble growth factors and the extracellular matrix. As focal adhesion kinase (FAK) integrates signals from both adhesion events and growth factor stimulation, we investigated its role in endothelial cell proliferation. Expression of a dominant-negative FAK protein, FAK-related nonkinase (FRNK), impaired phosphorylation of FAK and blocked DNA synthesis in response to multiple angiogenic stimuli. These results coincided with elevated cyclin-dependent kinase inhibitors (CDKIs) p21/Cip and p27/Kip, as a consequence of impaired degradation. FRNK inhibited the expression of Skp2, an F-box protein that targets CDKIs, by inhibiting mitogen-induced mRNA. The FAK-regulated degradation of p27/Kip was Skp2 dependent, while levels of p21/Cip were regulated independent of Skp2. Skp2 is required for endothelial cell proliferation as a consequence of degrading p27. Finally, knockdown of both p21 and p27 in FRNK-expressing cells completely restored mitogen-induced endothelial cell proliferation. These data demonstrate a critical role for FAK in the regulation of CDKIs through two independent mechanisms: Skp2 dependent and Skp2 independent. They also provide important insights into the requirement of focal adhesion kinase for normal vascular development and reveal novel regulatory control points for angiogenesis.     

Stimulation of phosphatidylcholine hydrolysis, diacylglycerol release, and arachidonic acid production by oncogenic ras is a consequence of protein kinase C activation

Swiss-3T3 cells were scrape-loaded with oncogenically activated p21ras protein. 10-20 min after introducing Val12p21ras into the cell, diacylglycerol levels were increased, but levels of inositol phosphates were unaltered. However, cellular choline and phosphocholine levels were increased with a similar time course to that observed for diacylglycerol production, suggesting that ras increases phosphatidylcholine turnover but not phosphatidylinositol turnover. Down-regulation of protein kinase C (by prolonged exposure to phorbol esters prior to scrape loading) blocked the ability of ras protein to elevate the levels of diacylglycerol, choline, and phosphocholine. Oncogenic ras can, therefore, cause a substantial increase in diacylglycerol (which correlates with increased phosphatidylcholine breakdown) in a protein kinase C-dependent fashion. Val12p21ras also increased arachidonic acid release, which was also dependent on protein kinase C activation. Induction of DNA synthesis by oncogenic ras was unaffected by inhibitors of prostaglandin synthesis, indicating that conversion of the released arachidonic acid to various prostaglandins is not required for stimulation of DNA synthesis by ras. We suggest that ras rapidly activates protein kinase C, which in turn activates a number of cellular signalling systems, leading to a sustained increase in diacylglycerol levels. This elevation of diacylglycerol could sustain protein kinase C activation over the 12-15 h required for initiation of DNA synthesis.     

The calcium‐sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium‐sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3‐phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H‐89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.     

Stress-stimulated mitogen-activated protein kinases control the stability and activity of the Cdt1 DNA replication licensing factor

DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G(1) phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G(2) phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitin-mediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4(Cdt2) targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G(2) promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.