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The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity. [ABSTRACT FROM AUTHOR]

Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phusphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21-/- cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on YI5 and phosphorylation on T161 promotes Cdc2 binding to the p21-eyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition. [ABSTRACT FROM AUTHOR]

In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules. [ABSTRACT FROM AUTHOR]

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that them TORC1 pathway and the oncogenic Ras/Raf/MEK/extra cellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote fromthe N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways. [ABSTRACT FROM AUTHOR]

One potentially important mechanism for regulating class Ia phosphoinositide 3-kinase (PI 3-kinase) activity is autophosphorylation of the p85α adapter subunit on Ser608 by the intrinsic protein kinase activity of the p110 catalytic subunit, as this downregulates the lipid kinase activity in vitro. Here we investigate whether this phosphorylation can occur in vivo. We find that p110α phosphorylates p85α Ser608 in vivo with significant stoichiometry. However, p110β is far less efficient at phosphorylating p85α Ser608, identifying a potential difference in the mechanisms by which these two isoforms are regulated. The p85α Ser608 phosphorylation was increased by treatment with insulin, platelet-derived growth factor, and the phosphatase inhibitor okadaic acid. The functional effects of this phosphorylation are highlighted by mutation of Ser608, which results in reduced lipid kinase activity and reduced association of the p110α catalytic subunit with p85α. The importance of this phosphorylation was further highlighted by the finding that autophosphorylation on Ser608 was impaired, while lipid kinase activity was increased, in a p85α mutant recently discovered in human tumors. These results provide the first evidence that phosphorylation of Set608 plays a role as a shutoff switch in growth factor signaling and contributes to the differences in functional properties of different PI 3-kinase isoforms in vivo. [ABSTRACT FROM AUTHOR]

In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt[sup S]) and long-period (dbt[sup L]) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt[sup S] and dbt[sup L] mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT[sup S], and DBTL proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt[sup S] and dbt[sup L] mutations reduced the CKI-7-sensitive kinase activity of an orthologons Xenopus laevis CKIδ expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt[sup S] and dbt[sup L] mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes. [ABSTRACT FROM AUTHOR]

Examines the role of Elm1 kinase activity inhibition in cell growth and cell division. Importance of Elm1 kinase activity in the proper organization of septins during G[sub 1]; Effect of inhibition of Elm1 kinase activity after bud emergence on mitotic delay; Implications of elm1 function loss; Possible functions of elm1 during G[sub 1] and G[sub 2]/M.

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-κB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-κB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncogenic mutant of TPL-2 is not affected by p105. Thus, in addition to its role as a precursor for p50 and cytoplasmic inhibitor of NF-κB, p105 is a negative regulator of TPL-2. Insensitivity of C-terminally truncated TPL-2 to this regulatory mechanism is likely to contribute to its ability to transform cells. [ABSTRACT FROM AUTHOR]

The p38 mitogen-activated protein kinase (MAPK) pathway is required for differentiation of skeletal myoblasts, but how the pathway is activated during this process is not well understood. One mechanism involves the cell surface receptor Cdo (also known as Cdon), which binds to Bnip-2 and JLP, scaffold proteins for Cdc42 and p38, respectively; formation of these complexes results in Bnip-2/Cdc42-dependent activation of p38. It has been reported that the tyrosine kinase Abl promotes myogenic differentiation in a manner dependent on its cytoplasmic localization, but the cytoplasmic signaling proteins with which it interacts to achieve this effect are unidentified. We report that Abl associates with both Cdo and JLP during myoblast differentiation. Abl binds a proline-rich motif in Cdo via its SH3 domain, and these regions of Abl and Cdo are required for their promyogenic effects. Cdo is important for full Abl kinase activity, and Abl is necessary for full activation of p38 MAPK, during myogenic differentiation. As seen with myoblasts depleted of Cdo, the diminished differentiation displayed by Abl-depleted cells is rescued by the expression of an activated form of the immediate upstream p38-activating kinase MAPK kinase 6. Abl's promyogenic effect is therefore linked to a multiprotein cell surface complex that regulates differentiation-dependent p38 activation. [ABSTRACT FROM AUTHOR]

The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. We describe a transgenic Drosophila model which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes. [ABSTRACT FROM AUTHOR]

Our studies examining the role of the cell cycle-regulated kinase cyclin A/Cdk2 in progesterone receptor (PR) action have demonstrated that cyclin-dependent kinase activity is required for PR function and that cyclin A/Cdk2 functions as a PR coactivator. Although Cdk2 can phosphorylate PR, elimination of these phosphorylation sites has little effect on the ability of cyclin A/Cdk2 to stimulate PR activity. PR interacts with cyclin A and recruits cyclin A/Cdk2 to progestin-responsive promoters, stimulating transcription. Inhibition of Cdk2 activity abolishes progesterone-dependent activation of PR target genes in part through inhibition of PR-dependent recruitment of steroid receptor coactivator 1 (SRC-1) and subsequent histone H4 acetylation at the target promoter. In vitro studies revealed that the interaction between SRC-1 and PR is dependent upon phosphorylation of SRC-1. This heretofore-unknown mechanism provides a potential means for integrating the regulation of PR activity with cell cycle progression. Moreover, the ability of PR to recruit cyclin A/Cdk2 to target promoters provides locally elevated levels of kinase, which can preferentially facilitate phosphorylation-dependent interactions and enzymatic activities of coactivators at the target promoter. [ABSTRACT FROM AUTHOR]

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4K1361R). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKKK1361R embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4K1361R embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4K1361R fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofiuorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not uitrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-ornothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts. [ABSTRACT FROM AUTHOR]

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G1. Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G0/G1. Here we show that RACK1 overexpression induces a partial G1 arrest by suppressing Src activity at the G1 checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G1/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G1/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G1 that works via an endogenous inhibitor of the Src kinase. [ABSTRACT FROM AUTHOR]

We recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G[sub 1] phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of α5β1 integrin. Mechanistically, α5β1 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G[sub 1] phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase led to a more rapid progression through G[sub 1] phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21[sup cip1] and p27[sup kip1], were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G[sub 1] phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G[sub 1] phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1. [ABSTRACT FROM AUTHOR]

Posttranslational modifications of histone proteins play important roles in the modulation of gene expression. The Saccharomyces cerevisiae (yeast) 2-MDa SAGA (Spt-Ada-Gcn5) complex, a well- studied multisubunit histone modifier, regulates gene expression through Gcn5-mediated histone acetylation and Ubp8-mediated histone deubiquitination. Using a proteomics approach, we determined that the SAGA complex also deubiquitinates nonhistone proteins, including Snf1, an AMP-activated kinase. Ubp8-mediated deubiquitination of Snf1 affects the stability and phosphorylation state of Snf1, thereby affecting Snf1 kinase activity. Others have reported that Gal83 is phosphorylated by Snf1, and we found that deletion of UBP8 causes decreased phosphorylation of Gal83, which is consistent with the effects of Ubp8 loss on Snf1 kinase functions. Overall, our data indicate that SAGA modulates the posttranslational modifications of Snf1 in order to fine-tune gene expression levels. [ABSTRACT FROM AUTHOR]

Myristoylation is critical for membrane association of Src kinases, but a role for myristate in regulating other aspects of Src biology has not been explored. In the c-Abl tyrosine kinase, myristate binds within a hydrophobic pocket at the base of the kinase domain and latches the protein into an autoinhibitory conformation. A similar pocket has been predicted to exist in c-Src, raising the possibility that Src might also be regulated by myristoylation. Here we show that in contrast to the case for c-Abl, myristoylation exerts a positive effect on c-Src kinase activity. We also demonstrate that myristoylation and membrane binding regulate c-Src ubiquitination and degradation. Nonmyristoylated c-Src exhibited reduced kinase activity but had enhanced stability compared to myristoylated c-Src. We then mutated critical residues in the predicted myristate binding pocket of c-Src. Mutation of L360 and/or E486 had no effect on c-Src membrane binding or localization. However, constructs containing a T456A mutation were partially released from the membrane, suggesting that mutagenesis could induce c-Src to undergo an artificial myristoyl switch. All of the pocket mutants exhibited decreased kinase activity. We concluded that myristoylation and the pocket residues regulate c-Src, but in a manner very different from that for c-Abl. [ABSTRACT FROM AUTHOR]

The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-lysine 3016 demonstrate rapid (within 5 min) in vivo acetylation of ATM following exposure to bleomycin. Furthermore, lysine 3016 of ATM is a substrate in vitro for the Tip60 histone acetyltransferase. Mutation of lysine 3016 does not affect unstimulated ATM kinase activity but does abolish upregulation of ATM's kinase activity by DNA damage, inhibits the conversion of inactive ATM dimers to active ATM monomers, and prevents the ATM-dependent phosphorylation of the p53 and chk2 proteins. These results are consistent with a model in which acetylation of lysine 3016 in the FATC domain of ATM activates the kinase activity of ATM. The acetylation of ATM on lysine 3016 by Tip60 is therefore a key step linking the detection of DNA damage and the activation of ATM kinase activity. [ABSTRACT FROM AUTHOR]

A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBTK/R) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBTK/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered "set point" for period length that is not altered by overexpression. Overexpression of the DBTK/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods. [ABSTRACT FROM AUTHOR]

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) resident PKR-like kinase (PERK) is necessary for Akt activation in response to ER stress. We demonstrate that PERK harbors intrinsic lipid kinase, favoring diacylglycerol (DAG) as a substrate and generating phosphatidic acid (PA). This activity of PERK correlates with activation of mTOR and phosphorylation of Akt on Ser473. PERK lipid kinase activity is regulated in a phosphatidylinositol 3-kinase (PI3K) p85α-dependent manner. Moreover, PERK activity is essential during adipocyte differentiation. Because PA and Akt regulate many cellular functions, including cellular survival, proliferation, migratory responses, and metabolic adaptation, our findings suggest that PERK has a more extensive role in insulin signaling, insulin resistance, obesity, and tumorigenesis than previously thought. [ABSTRACT FROM AUTHOR]

The article presents a research that investigates the role of mitogen-activated protein (MAP) in the regulation of kinase extracellular signal-regulated kinase 5 (ERK5) and cell proliferation.

Like phosphorylation, acetylation of lysine residues within a protein is considered a biologically relevant modification that controls the activity of target proteins. During stress of cells, massive protein acetylation takes place. Here, we show that p38 mitogen-activated protein kinase (MAPK), which controls many biological functions during stress, is reversibly acetylated by PCAF/p300 and HDAC3. We identified two acetylated lysine residues, K152 and K53, located in the substrate binding domain and in the ATP-binding pocket of p38, respectively. Acetylation of lysine 53 enhanced the activity of p38 by increasing its affinity for ATP binding. The enhanced acetylation and activation of p38 were found to be in parallel with reduced intracellular ATP levels in cardiomyocytes under stress, as well as in vivo models of cardiac hypertrophy. Thus, our data show, for the first time, that p38 activity is critically regulated by, in addition to phosphorylation, reversible acetylation of a lysine residue, which is conserved in other kinases, implying the possibility of a similar mechanism regulating their activity. [ABSTRACT FROM AUTHOR]

How a cell chooses between nonhomologous end joining (NHEJ) and homologous recombination (HR) to repair a double-strand break (DSB) is a central and largely unanswered question. Although there is evidence of competition between HR and NHEJ, because of the DNA-dependent protein kinase (DNA-PK)'s cellular abundance, it seems that there must be more to the repair pathway choice than direct competition. Both a mutational approach and chemical inhibition were utilized to address how DNA-PK affects HR. We find that DNA-PK's ability to repress HR is both titratable and entirely dependent on its enzymatic activity. Still, although requisite, robust enzymatic activity is not sufficient to inhibit HR. Emerging data (including the data presented here) document the functional complexities of DNA-PK's extensive phosphorylations that likely occur on more than 40 sites. Even more, we show here that certain phosphorylations of the DNA-PK large catalytic subunit (DNA-PKcs) clearly promote HR while inhibiting NHEJ, and we conclude that the phosphorylation status of DNA-PK impacts how a cell chooses to repair a DSB. [ABSTRACT FROM AUTHOR]

Promoter clearance and transcriptional processivity in eukaryotic cells are fundamentally regulated by the phosphorylation of the carboxy-terminal domain of RNA polymerase II (RNAPII). One of the kinases that essentially performs this function is P-TEFb (positive transcription elongation factor b), which is composed of cyclin-dependent kinase 9 (CDK9) associated with members of the cyclin T family. Here we show that cellular GCN5 and P/CAF, members of the GCN5-related N-acetyltransferase family of histone acetyltransferases, regulate CDK9 function by specifically acetylating the catalytic core of the enzyme and, in particular, a lysine that is essential for ATP coordination and the phosphotransfer reaction. Acetylation markedly reduces both the kinase function and transcriptional activity of P-TEFb. In contrast to unmodified CDK9, the acetylated fraction of the enzyme is specifically found in the insoluble nuclear matrix compartment. Acetylated CDK9 associates with the transcriptionally silent human immunodeficiency virus type 1 provirus; upon transcriptional activation, it is replaced by the unmodified form, which is involved in the elongating phase of transcription marked by Ser2-phosphorylated RNAPII. Given the conservation of the CDK9 acetylated residues in the catalytic task of virtually all CDK proteins, we anticipate that this mechanism of regulation might play a broader role in controlling the function of other members of this kinase family. [ABSTRACT FROM AUTHOR]

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation. [ABSTRACT FROM AUTHOR]

To investigate if intracellular glycerol content plays a role in the regulation of insulin secretion in pancreatic β cells, we studied the expression of the glycerol channels, or aquaglyceroporins, encoded by the aquaporin 3 (Aqp3), Aqp7, and Aqp9 genes in mouse islets. We found expression of Aqp7 only, not that of Aqp3 or Aqp9, in the endocrine pancreas at both the mRNA (by reverse transcription-PCR) and protein (by immunohistochemistry) levels. Immunohistochemistry revealed a complete overlap between insulin and Aqp7 immunostaining in the pancreatic islet. Inactivation of Aqp7 by gene targeting produced viable and healthy mice. Aqp7-/- mice harbored an increased intraislet glycerol concentration with a concomitant increase of the glycerol kinase transcript level and enzyme activity. The islet triglyceride content in the Aqp7-/- mice was also increased compared to that in the Aqp7+/+ mice. Interestingly, Aqp7-/- mice displayed reduced β-cell mass and insulin content but increased insulin-1 and insulin-2 mRNAs. The reduction of β-cell mass in Aqp7-/- mice can be explained at least in part by a reduction in cell proliferation through protein kinase C and the c-myc cascade, with a reduction in the transcript levels of these two genes. Concomitantly, there was a decreased rate of apoptosis, as reflected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase 3 and Bax expression in Aqp7-/- mice. Compared with Aqp7+/+ islets, islets isolated from Aqp7-/- mice secreted insulin at a higher rate under basal low-glucose conditions and on exposure to a high (450 mg/dl) glucose concentration. Aqp7-/- mice exhibited normal fasting blood glucose levels but elevated blood insulin levels. Their plasma glucose response to an intraperitoneal (i.p.) glucose tolerance test was normal, but their plasma insulin concentrations were higher than those of wild-type mice during the 2-h test. An i.p. insulin tolerance test showed similar plasma glucose lowering in Aqp7-/- and Aqp7+/+ mice, with no evidence of insulin resistance. In conclusion, we found that pancreatic β cells express AQP7, which appears to be a key regulator of intraislet glycerol content as well as insulin production and secretion. [ABSTRACT FROM AUTHOR]

The physiological functions of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus (Mapk6lacZ) showed that inactivation of ERK3 in Mapk6lacZ mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically inactive (Mapk6KD) or conditional (Mapk6Δ) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile, and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is necessary for optimal postnatal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that the expression of several Mapk6 neighboring genes is deregulated in Mapk6lacZ mice but not in Mapk6KD or Mapk6Δ mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are responsible for the perinatal lethality phenotype of Mapk6lacZ mutant mice. [ABSTRACT FROM AUTHOR]

Cullin-4 ubiquitin ligase (CRL4) complexes are differentially composed and highly dynamic protein assemblies that control many biological processes, including the global genome nucleotide excision repair (GG-NER) pathway. Here, we identified the kinase mitogen-activated protein kinase kinase kinase 1 (MEKK1) as a novel constitutive interactor of a cytosolic CRL4 complex that disassembles after DNA damage due to the caspase-mediated cleavage of MEKK1. The kinase activity of MEKK1 was important to trigger autoubiquitination of the CRL4 complex by K48- and K63-linked ubiquitin chains. MEKK1 knockdown prohibited DNA damage-induced degradation of the CRL4 component DNA-damage binding protein 2 (DDB2) and the CRL4 substrate p21 and also cell recovery and survival. A ubiquitin replacement strategy revealed a contribution of K63-branched ubiquitin chains for DNA damage-induced DDB2/p21 decay, cell cycle regulation, and cell survival. These data might also have implications for cancer, as frequently occurring mutations of MEKK1 might have an impact on genome stability and the therapeutic efficacy of CRL4-dependent immunomodulatory drugs such as thalidomide derivatives. [ABSTRACT FROM AUTHOR]

Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) is an emerging highly pathogenic phlebovirus. The syndrome is characterized by the substantial production of inflammatory cytokines and chemokines, described as a cytokine storm, which correlates with multiorgan failure and high mortality. SFSTV nonstructural (NSs) protein was suggested to mediate the pathogenesis by inhibiting antiviral interferon signaling in the host. However, whether SFTSV NSs protein mediates the induction of a fatal cytokine storm remains unaddressed. We demonstrated that SFTSV NSs promotes the hyperinduction of cytokine/chemokine genes in vitro, reminiscent of a cytokine storm. Using gene deletion and pharmacological intervention, we found that the induced cytokine storm is driven by the transcription factor NF-κB. Our investigation revealed that TANK-binding kinase 1 (TBK1) suppresses NF-κB signaling and cytokine/chemokine induction in a kinase activity-dependent manner and that NSs sequesters TBK1 to prevent it from suppressing NF-κB, thereby promoting the activation of NF-κB and its target cytokine/chemokine genes. Of note, NF-κB inhibition suppressed the induction of proinflammatory cytokines in SFTSV-infected type I interferon (IFN-I) receptor 1-deficient (Ifnar1-/-) mice. These findings establish the essential role of NSs in SFTS pathogenesis and suggest NF-κB as a possible therapeutic target. [ABSTRACT FROM AUTHOR]

Cell polarity is essential for various cellular functions during both proliferative and developmental stages, and it displays dynamic alterations in response to intracellular and extracellular cues. However, the molecular mechanisms underlying spatiotemporal control of polarity transition are poorly understood. Here, we show that fission yeast Cki3 (a casein kinase 1γ homolog) is a critical regulator to ensure persistent monopolar growth during S phase. Unlike the wild type, cki3 mutant cells undergo bipolar growth when S phase is blocked, a condition known to delay transition from monopolar to bipolar growth (termed NETO [new end takeoff]). Consistent with this role, Cki3 kinase activity is substantially increased, and cells lose their viability in the absence of Cki3 upon an S-phase block. Cki3 acts downstream of the checkpoint kinase Cds1/Chk2 and calcineurin, and the latter physically interacts with Cki3. Autophosphorylation in the C terminus is inhibitory toward Cki3 kinase activity, and calcineurin is responsible for its dephosphorylation. Cki3 localizes to the plasma membrane, and this localization requires the palmitoyltransferase complex Erf2-Erf4. Membrane localization is needed not only for proper NETO timing but also for Cki3 kinase activity. We propose that Cki3 acts as a critical inhibitor of cell polarity transition under S-phase arrest. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity. [ABSTRACT FROM AUTHOR]

JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. [ABSTRACT FROM AUTHOR]

Often considered to be a "dead" kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation is shown to markedly upregulate kinase activity in erbB3 immunoprecipitates. Intact, activated erbB3 phosphorylates tyrosine sites in an exogenous peptide substrate, and this activity is abolished by mutagenesis of lysine 723 in the catalytic domain. Enhanced erbB3 kinase activity is linked to heterointeractions with catalytically active erbB2, since it is largely blocked in cells pretreated with lapatinib or pertuzumab. erbB2 activation of erbB3 is not dependent on equal surface levels of these receptors, since it occurs even in erbB3-transfected CHO cells with disproportionally small amounts of erbB2. We tested a model in which transient erbB3/erbB2 heterointeractions set the stage for erbB3 homodimers to be signaling competent. erbB3 homoand heterodimerization events were captured in real time on live cells using single-particle tracking of quantum dot probes bound to ligand or hemagglutinin tags on recombinant receptors. [ABSTRACT FROM AUTHOR]

Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1-/- and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1-/- mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS).We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NFκ-B-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling. [ABSTRACT FROM AUTHOR]

V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs3A) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpinsealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs. [ABSTRACT FROM AUTHOR]

Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed the "ER stress", activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog Kin2 in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity37 regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an auto-inhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for the UPR function. We also show that the functional mini Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that trans-phosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for the full kinase activity. Collectively, our data suggest that, during the ER stress, the Kin kinase domain is released from its auto-inhibitory KA1 domain and is activated by trans-phosphorylation. [ABSTRACT FROM AUTHOR]

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 G1 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 G2 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 CRL4Cdt2 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 G2 promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete. [ABSTRACT FROM AUTHOR]

TEL1 is important in Saccharomyces cerevisiae telomere maintenance, and its kinase activity is required. Tel1p associates with telomeres in vivo, is enriched at short telomeres, and enhances the binding of telomerase components to short telomeres. However, it is unclear how the kinase activity and telomere association contribute to Tel1p's overall function in telomere length maintenance. To investigate this question, we generated a set of single point mutants and a double point mutant (tel1KD) of Tel1p that were kinase deficient and two Xrs2p mutants that failed to bind Tel1p. Using these separation-of-function alleles in a de novo telomere elongation assay, we found, surprisingly, that the tel1KD allele and xrs2 C-terminal mutants were both partially functional. Combining the tel1KD and xrs2 C-terminal mutants had an additive effect and resembled the TEL1 null (tel1Δ) phenotype. These data indicate that Tel1p has two separate functions in telomere maintenance and that the Xrs2p-dependent recruitment of Tel1p to telomeres plays an important role even in the absence of its kinase activity. [ABSTRACT FROM AUTHOR]

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12—but not Med13—to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex—unlike the recombinant submodule—is unable to phosphorylate the Pol II CTD. [ABSTRACT FROM AUTHOR]

Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs. [ABSTRACT FROM AUTHOR]

Ctk1 is a Saccharomyces cerevisiae cyclin-dependent protein kinase (CDK) that assembles with Ctk2 and Ctk3 to form an active protein kinase complex, CTDK-L CTDK-I phosphorylates Ser2 within the RNA polymerase II C-terminal domain, an activity that is required for efficient transcriptional elongation and 3' RNA processing. Ctk1 contains a conserved T loop, which undergoes activating phosphorylation in other CDKs. We show that Ctk1 is phosphorylated on Thr-338 within the T loop. Mutation of this residue abolished Ctk1 kinase activity in vitro and resulted in a cold-sensitive phenotype. As with other yeast CDKs undergoing T-loop phosphorylation, Ctk1 phosphorylation on Thr-338 was dependent on the Cak1 protein kinase. Ctk1 isolated from cak1Δ cells was unphosphorylated and exhibited low protein kinase activity. Moreover, Cak1 directly phosphorylated Ctk1 in vitro. Unlike wild-type cells, cells expressing Ctk1T338A delayed growth at early stationary phase, did not show the increase in Ser2 phosphorylation that normally accompanies the transition from rapid growth to stationary phase, and had compromised transcriptional activation of two stationary- phase genes, CTT1 and SF11. Therefore, Ctkl phosphorylation on Thr-338 is carried out by Cak1 and is required for normal gene transcription during the transition into stationary phase. [ABSTRACT FROM AUTHOR]

Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns (3)P-to-PtdIns (3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endo-membrane homeostasis in mammalian cells. [ABSTRACT FROM AUTHOR]

The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-κB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis, p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IκB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-κB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade. [ABSTRACT FROM AUTHOR]

Germinal center kinase (GCK), a member of the Ste20 family, selectively activates the Jun N-terminal kinase (JNK) group of mitogen-activated protein kinases. Here, we show that endogenous GCK is activated by polyinosine-polycytidine [poly(IC)] and iipopolysaccharides (LPS), lipid A, interleukin-1 (IL-1), and engagement of CD40, all agonists that require TRAF6 for JNK activation. RNA interference experiments indicate that GCK is required for the maximal activation of JNK by LPS, lipid A, poly(IC), and, to a lesser extent, IL-1 and engagement of CD40. GCK is ubiquitinated in situ and stabilized by inhibitors of the proteasome, indicating that GCK is subject to proteasomal turnover. GCK is constitutively active, and the kinase activity of GCK is required for GCK ubiquitination. Agonist activation of GCK involves the TRAF6-dependent transient stabilization of the GCK polypeptide rather than an increase in intrinsic kinase activity. Our results identify a physiologic function and unexpected mode of regulation for GCK. [ABSTRACT FROM AUTHOR]

Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca2+, released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-γ and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcεRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcεRI triggering, enhanced sphingosine kinase activity was associated with FcεRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn-/- mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcεRI proximal event. [ABSTRACT FROM AUTHOR]

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2. [ABSTRACT FROM AUTHOR]

Activation of either the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt or the p38 mitogen-activated protein kinase (MAPK) signaling pathways accelerates myogenesis but only when the reciprocal pathway is functional. We therefore examined the hypothesis that cross-activation between these signaling cascades occurs to orchestrate myogenesis. We reveal a novel and reciprocal cross-talk and activation between the PI 3-kinase/Akt and p38 MAPK pathways that is essential for efficient myoblast differentiation. During myoblast differentia. tion, Akt kinase activity correlated with S473 but not T308 phosphorylation and occurred 24 h after p38 activation. Inhibition or activation of p38 with SB203580, dominant-negative p38, or MKK6EE regulated Akt kinase activity. Analysis of Akt isoforms revealed a specific increase in Akt2 protein levels that coincided with AktS473 phosphorylation during myogenesis and an enrichment of S473-phosphorylated Akt2. Akt2 promoter activity and protein levels were regulated by p38 activation, thus providing a mechanism for communication. Subsequent Akt activation by S473 phosphorylation was PI 3-kinase dependent and specific for Akt2 rather than Akt1. Complementary to p38-mediated transactivation of Akt, activation or inhibition of PI 3-kinase regulated p38 activity upstream of MKK6, demonstrating reciprocal communication and positive feedback characteristic of myogenic regulation. Our findings have identified novel communication between p38 MAPK and PI 3-kinase/Akt via Akt2. [ABSTRACT FROM AUTHOR]

The Sid2p-Mob1p kinase complex is an important component of the septation initiation network (SIN) in the fission yeast Schizosaccharomyces pombe. However, regulation of this complex is still elusive. Here we show that Mob1p is required not only for the subcellular localization of Sid2p but also for its kinase activity. We identified a region at the amino terminus of Sid2p that is required for Mob1p binding and spindle pole body (SPB) localization. Deletion of this region abolishes Mob1p binding and diminishes SPB localization, whereas this region alone is sufficient to associate with Mob1p and SPBs. We further show that a similar region of the N terminus of the Sid2p-related protein kinase Orb6p binds to the Mob1p-related protein Mob2p, suggesting that this may be a conserved mode of interaction for this family of kinases. Phosphorylation of Ser402 and especially Thr578 is important for Sid2p function. Sid2p with a mutation of Thr578 to Ala (T578A) can no longer rescue sid2-250 mutant cells, and this results in reduction of Mob1p binding. Sid2p mutants mimicking phosphorylation at this site (T578D and T578E) can rescue sid2-250 cells, enhance Sid2p kinase activity, and partially rescue growth defects of upstream sin mutants. Interestingly, Sid2p, but not Mob1p, is self-associated. Our experiments suggest that self-associated Sid2p is inactive. This self-association is mediated by a region that overlaps with Mob1p and SPB binding sites. Overexpression of Mobip is able to disrupt the self-association of Sid2p. Taken together, our results suggest that Sid2p kinase may utilize multiple modes of regulation including self-association, Mob1p binding, and phosphorylation to achieve its full activity at an appropriate time and place in the cell. [ABSTRACT FROM AUTHOR]