Regulation of minute virus of mice NS1 replicative functions by atypical PKClambda in vivo

Minute virus of mice NS1 protein is a multifunctional phosphoprotein endowed with a variety of enzymatic and regulatory activities necessary for progeny virus particle production. To regulate all of its different functions in the course of a viral infection, NS1 has been proposed to be modulated by posttranslational modifications, in particular, phosphorylation. Indeed, it was shown that the NS1 phosphorylation pattern is altered during the infectious cycle and that the biochemical profile of the protein is dependent on the phosphorylation state of the polypeptide. Moreover, in vitro approaches have identified members of the protein kinase C (PKC) family, in particular, atypical PKC, as regulators of viral DNA replication through the phosphorylation of NS1 residues T435 and S473, thereby activating the protein for DNA unwinding activities. In order to substantiate these findings in vivo, we produced NS1 in the presence of a dominant-negative PKClambda mutant and characterized the purified protein in vitro. The NS1 protein produced under these conditions was found to be only partially phosphorylated and as a consequence to be deficient for viral DNA replication. However, it could be rescued for this viral function by treatment with recombinant activated PKClambda. Our data clearly demonstrate that NS1 is a target for PKClambda phosphorylation in vivo and that this modification is essential for the helicase activity of the viral polypeptide. In addition, the phosphorylation of NS1 at residues T435 and S473 appeared to occur mainly in the nucleus, providing further evidence for the involvement of PKClambda which, unlike PKCzeta, accumulates in the nuclear compartment of infected cells.     

Initiation of minute virus of mice DNA replication is regulated at the level of origin unwinding by atypical protein kinase C phosphorylation of NS1

Minute virus of mice nonstructural protein NS1 is a multifunctional protein that is involved in many processes necessary for virus propagation. To perform its distinct activities in timely coordinated manner, NS1 was suggested to be regulated by posttranslational modifications, in particular phosphorylation. In fact, NS1 replicative functions are dependent on protein kinase C (PKC) phosphorylation, most likely due to alteration of the biochemical profile of the viral product as determined by comparing native NS1 with its dephosphorylated counterpart. Through the characterization of NS1 mutants at individual PKC consensus phosphorylation sites for their biochemical activities and nickase function, we were able to identify two target atypical PKC phosphorylation sites, T435 and S473, serving as regulatory elements for the initiation of viral DNA replication. Furthermore, by dissociating the energy-dependent helicase activity from the ATPase-independent trans esterification reaction using partially single-stranded substrates, we could demonstrate that atypical PKC regulation of NS1 nickase activity occurs at the level of origin unwinding prior to trans esterification.     

Novel PKCeta is required to activate replicative functions of the major nonstructural protein NS1 of minute virus of mice

The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKClambda) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKCeta phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKClambda for rolling circle replication. Moreover, this role of PKCeta was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKCeta mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically (32)P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCetaDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKCeta in the nuclear periphery, suggesting that besides being a target for PKCeta, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle.     

Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1^O) and the native (NS1^P) polypeptides. Biochemical analyses of replicative functions of NS1^O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]_2–3 was enhanced compared to that of NS1^P. In the presence of endogenous protein kinases found in replication extracts, NS1^O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1^O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1^O was incubated with protein kinase C.     

Roles of the Phosphorylation of Specific Serines and Threonines in the NS1 Protein of Human Influenza A Viruses

We demonstrate that phosphorylation of the NS1 protein of a human influenza A virus occurs not only at the threonine (T) at position 215 but also at serines (Ss), specifically at positions 42 and 48. By generating recombinant influenza A/Udorn/72 (Ud) viruses that encode mutant NS1 proteins, we determined the roles of these phosphorylations in virus replication. At position 215 only a T-to-A substitution attenuated replication, whereas other substitutions (T to E to mimic constitutive phosphorylation, T to N, and T to P, the amino acid in avian influenza A virus NS1 proteins) had no effect. We conclude that attenuation resulting from the T-to-A substitution at position 215 is attributable to a deleterious structural change in the NS1 protein that is not caused by other amino acid substitutions and that phosphorylation of T215 does not affect virus replication. At position 48 neither an S-to-A substitution nor an S-to-D substitution that mimics constitutive phosphorylation affected virus replication. In contrast, at position 42, an S-to-D, but not an S-to-A, substitution caused attenuation. The S-to-D substitution eliminates detectable double-stranded RNA binding by the NS1 protein, accounting for attenuation of virus replication. We show that protein kinase C α (PKCα) catalyzes S42 phosphorylation. Consequently, the only phosphorylation of the NS1 protein of this human influenza A virus that regulates its replication is S42 phosphorylation catalyzed by PKCα. In contrast, phosphorylation of Ts or Ss in the NS1 protein of the 2009 H1N1 pandemic virus was not detected, indicating that NS1 phosphorylation probably does not play any role in the replication of this virus.     

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.     

Protein kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine 81 phosphorylation site

The Ca(v)1.3 (alpha(1D)) variant of L-type Ca(2+) channels plays a vital role in the function of neuroendocrine and cardiovascular systems. In this article, we report on the molecular and functional basis of alpha(1D) Ca(2+) channel modulation by protein kinase C (PKC). Specifically, we show that the serine 81 (S81) phosphorylation site at the NH(2)-terminal region plays a critical role in alpha(1D) Ca(2+) channel modulation by PKC. The introduction of a negatively charged residue at position 81, by converting serine to aspartate, mimicked the PKC phosphorylation effect on alpha(1D) Ca(2+) channel. The modulation of alpha(1D) Ca(2+) channel by PKC was prevented by dialyzing cells with a 35-amino acid peptide mimicking the alpha(1D) NH(2)-terminal region comprising S81. In addition, the data revealed that only betaII- and epsilonPKC isozymes are implicated in this regulation. These novel findings have significant implications in the pathophysiology of alpha(1D) Ca(2+) channel and in the development of PKC isozyme-targeted therapeutics.     

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1–168) joined to an RNA helicase (residues 180–618) by an 11-amino acid linker (169–179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B₁₈NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173–183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ∼161° with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn²⁺ refined to a resolution of 2.2 Å. The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu¹⁷³ and Pro¹⁷⁴ or replacing Pro¹⁷⁴ with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro¹⁷⁶ to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication.     

Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

Protein phosphatase PP4C has been implicated in the DNA damage response (DDR), but its substrates in DDR remain largely unknown. We devised a novel proteomic strategy for systematic identification of proteins dephosphorylated by PP4C and identified KRAB-domain-associated protein 1 (KAP-1) as a substrate. Ionizing radiation leads to phosphorylation of KAP-1 at S824 (via ATM) and at S473 (via CHK2). A PP4C/R3β complex interacts with KAP-1 and silencing this complex leads to persistence of phospho-S824 and phospho-S473. We identify a new role for KAP-1 in DDR by showing that phosphorylation of S473 impacts the G2/M checkpoint. Depletion of PP4R3β or expression of the phosphomimetic KAP-1 S473 mutant (S473D) leads to a prolonged G2/M checkpoint. Phosphorylation of S824 is necessary for repair of heterochromatic DNA lesions and similar to cells expressing phosphomimetic KAP-1 S824 mutant (S824D), or PP4R3β-silenced cells, display prolonged relaxation of chromatin with release of chromatin remodelling protein CHD3. Our results define a new role for PP4-mediated dephosphorylation in the DDR, including the regulation of a previously undescribed function of KAP-1 in checkpoint response.     

Inhibition of insulin-induced glucose uptake by atypical protein kinase C isotype-specific interacting protein in 3T3-L1 adipocytes

Atypical protein kinase C (PKC) isotype-specific interacting protein (ASIP) specifically interacts with the atypical protein kinase C isozymes PKClambda and PKCzeta. ASIP and atypical PKC, as well as their Caenorhabditis elegans counterparts (PAR-3 and PKC-3, respectively), are thought to coordinately participate in intracellular signaling that contributes to the maintenance of cellular polarity and to the formation of junctional complexes. The potential role of ASIP in other cellular functions of atypical PKC was investigated by examining the effect of overexpression of ASIP on insulin-induced glucose uptake, previously shown to be mediated through PKClambda, in 3T3-L1 adipocytes. When overexpressed in these cells, which contain PKClambda but not PKCzeta, ASIP was co-immunoprecipitated with endogenous PKClambda but not with PKCepsilon or with Akt. The subcellular localization of PKClambda was also altered in cells overexpressing ASIP. Overexpression of ASIP inhibited insulin stimulation of both glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but it did not inhibit glucose uptake induced by either growth hormone or hyperosmolarity both of which promote glucose uptake in a PKClambda-independent manner. Moreover, glucose uptake stimulated by a constitutively active mutant of PKClambda, but not that induced by an active form of Akt, was inhibited by ASIP. Insulin-induced activation of PKClambda, but not that of phosphoinositide 3-kinase or Akt, was also inhibited by overexpression of ASIP. These data suggest that overexpression of ASIP inhibits insulin-induced glucose uptake by specifically interfering with signals transmitted through PKClambda.     

Modulation of Hepatitis C Virus Genome Encapsidation by Nonstructural Protein 4B

Hepatitis C Virus (HCV) NS4B protein has many roles in HCV genome replication. Recently, our laboratory (Q. Han, J. Aligo, D. Manna, K. Belton, S. V. Chintapalli, Y. Hong, R. L. Patterson, D. B. van Rossum, and K. V. Konan, J. Virol. 85:6464–6479, 2011) and others (D. M. Jones, A. H. Patel, P. Targett-Adams, and J. McLauchlan, J. Virol. 83:2163–2177, 2009; D. Paul, I. Romero-Brey, J. Gouttenoire, S. Stoitsova, J. Krijnse-Locker, D. Moradpour, and R. Bartenschlager, J. Virol. 85:6963–6976, 2011) have also reported NS4B''s function in postreplication steps. Indeed, replacement of the NS4B C-terminal domain (CTD) in the HCV JFH1 (genotype 2a [G2a]) genome with sequences from Con1 (G1b) or H77 (G1a) had a negligible impact on JFH1 genome replication but attenuated virus production. Since NS4B interacts weakly with the HCV genome, we postulated that NS4B regulates the function of host or virus proteins directly involved in HCV production. In this study, we demonstrate that the integrity of the JFH1 NS4B CTD is crucial for efficient JFH1 genome encapsidation. Further, two adaptive mutations (NS4B N216S and NS5A C465S) were identified, and introduction of these mutations into the chimera rescued virus production to various levels, suggesting a genetic interaction between the NS4B and NS5A proteins. Interestingly, cells infected with chimeric viruses displayed a markedly decreased NS5A hyperphosphorylation state (NS5A p58) relative to JFH1, and the adaptive mutations differentially rescued NS5A p58 formation. However, immunofluorescence staining indicated that the decrease in NS5A p58 did not alter NS5A colocalization with the core around lipid droplets (LDs), the site of JFH1 assembly, suggesting that NS5A fails to facilitate the transfer of HCV RNA to the capsid protein on LDs. Alternatively, NS4B''s function in HCV genome encapsidation may entail more than its regulation of the NS5A phosphorylation state.     

Effects of in vitro dephosphorylation on DNA-binding and DNA helicase activities of simian virus 40 large tumor antigen.

Simian virus 40 large T antigen is a phosphoprotein with two clusters of phosphorylation sites. Each cluster includes four serine residues and one threonine residue. In vitro treatment with intestinal alkaline phosphatase removes the phosphate groups from the serine but not from the threonine residues. Potato acid phosphatase additionally dephosphorylates the phosphothreonine (Thr-124) in the N-terminal cluster but does not attack the phosphothreonine in the C-terminal cluster (Thr-701). Two biochemical functions of untreated and partially dephosphorylated T antigen were assayed, namely, its specific DNA-binding property and its DNA helicase activity. After treatment with alkaline phosphatase, T antigen had a severalfold higher affinity for the specific binding sites in the viral genomic control region, in particular, for binding site II in the origin of replication. However, T antigen, when dephosphorylated by acid phosphatase, had DNA-binding properties similar to those of the untreated control. Neither alkaline nor acid dephosphorylation affected the DNA helicase activity of T antigen.     

Syndecan 4 regulation of PDK1-dependent Akt activation

The phosphatidylinositol 3 kinase (Pi3K)/Akt pathway is a major regulator of cell growth, proliferation, metabolism, survival, and angiogenesis. Despite extensive study, a thorough understanding of the modulation and regulation of this pathway has remained elusive. We have previously demonstrated that syndecan 4 (S4) regulates the intracellular localization of mTORC2, thus altering phosphorylation of Akt at serine473 (Ser473), one of two critical phosphorylation sites essential for the full activation of Akt [1]. Here we report that S4 also regulates the phosphorylation of Akt at threonine308 (Thr308), the second phosphorylation site required for the full Akt activation. A deletion of S4 resulted in lower levels of Thr308 phosphorylation both in vitro and in vivo. Furthermore, a deletion or knockdown of the S4 effector molecule PKCα led to a similar reduction in phosphorylation of Thr308 while overexpression of myristoylated PKCα rescued AktThr308 phosphorylation in endothelial cells lacking S4. Finally, PAK1/2 is also recruited to the rafts by the S4-PKCα complex and is required for AKT activation.     

Mechanism of inhibition of insulin-stimulated glucose transport by 4-bromocrotonic acid in 3T3-L1 adipocytes

We have demonstrated previously that 4-bromocrotonic acid (Br-C4) inhibited insulin-stimulated glucose transport by interfering with GLUT4 translocation. In the present study, we further examined the underlying mechanism involved. Since insulin-induced insulin receptor substrate-1-associated phosphatidylinositol (PI) 3-kinase activity was not altered by Br-C4, we determined and found insulin activation of protein kinase B (PKB) and protein kinase Clambda (PKClambda) were both inhibited. However, time-course studies showed that only the inhibition of PKB activation correlated with the inhibition of insulin-stimulated glucose transport. In concert, insulin-stimulated Ser(473/474) phosphorylation on PKB(alpha/beta) were similarly decreased by Br-C4. The finding that okadaic acid-stimulated glucose transport and PKClambda activity were both inhibited by Br-C4 suggested that the effect of Br-C4 on Ser(473/474) phosphorylation was not mediated by protein phosphatase 2A. Moreover, whereas Br-C4 nearly abolished insulin-stimulated integrin-linked kinase (ILK) activity, it only inhibited insulin-stimulated PKB activity by 20%, implying that ILK was not the major kinase for Ser(473/474) phosphorylation. Taken together, these results support the notion that PKB is involved in insulin-stimulated glucose transport. In addition, Br-C4 seems to inhibit insulin-stimulated glucose transport via inhibiting insulin activation of PKB, probably by interfering with insulin activation of an upstream kinase responsible for the phosphorylation of Ser(473/474) residue.     

Activation of a PKC is required for vanadate-induced phosphorylation of protein kinase B (Akt), but not p70S6k in mouse epidermal JB6 cells

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6K in a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (lambda, zeta) from the cytosol to the membrane, but had no effect on PKCalpha translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKClambda blocked Akt phosphorylation at Ser473 and Thr308, whereas it did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6k phosphorylation at Thr389 and Thr421/Ser424 and Akt phosphorylation at Thr308 occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6k were different.     

Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity.

A consensus sequence in parvovirus nonstructural protein NS1 has been predicted to be an ATP-binding domain associated with an ATPase and a DNA helicase activity. To investigate the function of NS1 in viral gene expression, a site-directed mutagenesis converting NS1 lysine 405 to serine in parvovirus H-1 was carried out by the polymerase chain reaction. As shown previously, a parvovirus genome containing a deleted NS1 gene was excised from a bacterial plasmid and replicated when a wild-type NS1 gene was provided in trans but failed to be excised and replicate when the mutant NS1 gene was supplied. Interestingly, the serine 405 mutation totally lost the activity of trans activation on the virus late promoter (P38) in a chloramphenicol acetyltransferase (CAT) assay and it lost evidence for cytotoxicity in two tumor cell lines (HeLa Gey and NB324K). The serine 405 NS1 protein was translocated normally to the nucleus. These results suggest that the NS1 lysine 405 of H-1 in its putative purine nucleotide-binding site is essential for viral DNA replication and that this domain may be involved in the regulation of the P38 promoter by an unknown mechanism. The loss of NS1 cytotoxicity on tumor cells suggests that NS1 expression is the major cause of cell killing by parvoviruses, which may facilitate further study of the mechanism of oncosuppression by parvoviruses.     

Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.     

Targeted deletion of protein kinase C lambda reveals a distribution of functions between the two atypical protein kinase C isoforms

Protein kinase C lambda (PKClambda) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCzeta. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKClambda and PKCzeta have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-kappaB. In addition, work from invertebrates shows that atypical PKC molecules play a role in embryo development and cell polarity. To determine the unique functions of PKClambda, mice deficient for PKClambda were generated by gene targeting. The ablation of PKClambda results in abnormalities early in gestation with lethality occurring by embryonic day 9. The role of PKClambda in cytokine-mediated cellular activation was studied by making mouse chimeras from PKClambda-deficient embryonic stem cells and C57BL/6 or Rag2-deficient blastocysts. Cell lines derived from these chimeric animals were then used to dissect the role of PKClambda in cytokine responses. Although the mutant cells exhibited alterations in actin stress fibers and focal adhesions, no other phenotypic differences were noted. Contrary to experiments using dominant interfering forms of PKClambda, mutant cells responded normally to TNF, serum, epidermal growth factor, IL-1, and LPS. In addition, no abnormalities were found in T cell development or T cell activation. These data establish that, in vertebrates, the two disparate functions of atypical PKC molecules have been segregated such that PKCzeta mediates signal transduction of the innate immune system and PKClambda is essential for early embryogenesis.     

Protein Phosphatase 2A and Cdc7 Kinase Regulate the DNA Unwinding Element-binding Protein in Replication Initiation

The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2–7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2–7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.