The signaling axis atypical protein kinase C λ/ι-Satb2 mediates leukemic transformation of B-cell progenitors.

Epigenetically regulated transcriptional plasticity has been proposed as a mechanism of differentiation arrest and resistance to therapy. BCR-ABL leukemias result from leukemic stem cell/progenitor transformation and represent an opportunity to identify epigenetic progress contributing to lineage leukemogenesis. Primary human and murine BCR-ABL+ leukemic progenitors have increased activation of Cdc42 and the downstream atypical protein kinase C (aPKC). While the isoform aPKCζ behaves as a leukemic suppressor, aPKCλ/ι is critically required for oncogenic progenitor proliferation, survival, and B-cell differentiation arrest, but not for normal B-cell lineage differentiation. In vitro and in vivo B-cell transformation by BCR-ABL requires the downregulation of key genes in the B-cell differentiation program through an aPKC λ/ι-Erk dependent Etv5/Satb2 chromatin repressive signaling complex. Genetic or pharmacological targeting of aPKC impairs human oncogenic addicted leukemias. Therefore, the aPKCλ/ι-SATB2 signaling cascade is required for leukemic BCR-ABL+ B-cell progenitor transformation and is amenable to non-tyrosine kinase inhibition.

Of the atypical PKCs, Par-4 and p62: recent understandings of the biology and pathology of a PB1-dominated complex.

The recent identification of a novel protein-protein interaction module, termed PB1, in critical signaling molecules such as p62 (also known as sequestosome1), the atypical PKCs, and Par-6, has unveiled the existence of a new set of signaling complexes, which can be central to several biological processes from development to cancer. In this review, we will discuss the most recent advances on the role that the different components of these complexes have in vivo and that are relevant to human disease. In particular, we will review what we are learning from new data from knockout mice, and the indications from human mutations on the real role of these proteins in the physiology and biology of human diseases. The role that PKCzeta, PKClambda/iota, and Par-4 have in lung and prostate cancer in vivo and in humans will be extensively covered in this article, as will the multifunctional role of p62 as a novel hub in cell signaling during cancer and inflammation, and the mechanistic details and controversial data published on its potential role in aggregate formation and signaling. All this published information is shedding new light on the proposed pathological implications of these PB1-regulators in disease and shows their important role in cell physiology.

PKCzeta at the crossroad of NF-kappaB and Jak1/Stat6 signaling pathways.

The atypical protein kinase C (PKC) isoforms (aPKC) have been implicated in the regulation of a number of essential signaling events. Early studies using dominant-negative mutants suggested that they are important intermediaries in the activation of the canonical nuclear factor (NF)-kappaB pathway. More recent data using knockout mice genetically demonstrate that in fact the PKCzeta isoform is essential for the adequate activation of this cascade both upstream and downstream the IkappaB kinase complex. In this review, we summarize the mechanistic details whereby the aPKC pathway regulates important cellular functions and how this is achieved by the ability of these kinases to interact with different protein regulators and adapters, as well as to impinge in NF-kappaB-independent signaling cascades such as the Janus kinase-1/signal transducer and activator of transcription 6 system, which plays a critical role in T-cell-mediated hepatitis and asthma.

Targeted disruption of the zetaPKC gene results in the impairment of the NF-kappaB pathway.

Here we have addressed the role that zetaPKC plays in NF-kappaB activation using mice in which this kinase was inactivated by homologous recombination. These mice, although grossly normal, showed phenotypic alterations in secondary lymphoid organs reminiscent of those of the TNF receptor-1 and of the lymphotoxin-beta receptor gene-deficient mice. The lack of zetaPKC in embryonic fibroblasts (EFs) severely impairs kappaB-dependent transcriptional activity as well as cytokine-induced phosphorylation of p65. Also, a cytokine-inducible interaction of zetaPKC with p65 was detected which requires the previous degradation of IkappaB. Although in zetaPKC-/- EFs this kinase is not necessary for IKK activation, in lung, which abundantly expresses zetaPKC, IKK activation is inhibited.

The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor.

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor kappaB (NF-kappaB). Here we show that the atypical protein kinase C-interacting protein, p62, which binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. We demonstrate the formation of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, we demonstrated that p62 serves as a scaffold for activation of the NF-kappaB pathway, which mediates NGF survival and differentiation responses.

MEK5, a new target of the atypical protein kinase C isoforms in mitogenic signaling.

The MEK5-extracellular signal-regulated kinase (ERK5) tandem is a novel mitogen-activated protein kinase cassette critically involved in mitogenic activation by the epidermal growth factor (EGF). The atypical protein kinase C isoforms (aPKCs) have been shown to be required for cell growth and proliferation and have been reported to interact with the adapter protein p62 through a short stretch of acidic amino acids termed the aPKC interaction domain. This region is also present in MEK5, suggesting that it may be an aPKC-binding partner. Here we demonstrate that the aPKCs interact in an EGF-inducible manner with MEK5 and that this interaction is required and sufficient for the activation of MEK5 in response to EGF. Consistent with the role of the aPKCs in the MEK5-ERK5 pathway, we show that zetaPKC and lambda/iotaPKC activate the Jun promoter through the MEF2C element, a well-established target of ERK5. From all these results, we conclude that MEK5 is a critical target of the aPKCs during mitogenic signaling.

The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.

Liver injury during cholestasis reflects a balance between the effects of toxic and nontoxic bile acids. However, the critical distinction between a toxic and nontoxic bile acid remains subtle and unclear. For example, the glycine conjugate of chenodeoxycholate (GCDC) induces hepatocyte apoptosis, whereas the taurine conjugate (TCDC) does not. We hypothesized that the dissimilar cellular responses may reflect differential activation of a phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. In the bile acid-transporting McNtcp.24 rat hepatoma cell line, TCDC, but not GCDC, stimulated PI3K activity. Consistent with this observation, inhibition of PI3K rendered TCDC cytotoxic, and constitutive activation of PI3K rendered GCDC nontoxic. Both Akt and the atypical protein kinase C isoform zeta (PKCzeta) have been implicated in PI3K-dependent survival signaling. However, TCDC activated PKCzeta, but not Akt. Moreover, inhibition of PKCzeta converted TCDC into a cytotoxic agent, whereas overexpression of wild-type PKCzeta blocked GCDC-induced apoptosis. We also demonstrate that TCDC activated nuclear factor kappaB (NF-kappaB) in a PI3K- and PKCzeta-dependent manner. Moreover, inhibition of NF-kappaB by an IkappaB super-repressor rendered TCDC cytotoxic, suggesting that NF-kappaB is also necessary to prevent the cytotoxic effects of TCDC. Collectively, these data suggest that some hydrophobic bile acids such as TCDC activate PI3K-dependent survival pathways, which prevent their otherwise inherent toxicity.

The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation.

Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)-rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK(3/6)-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors.

The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway.

The atypical protein kinase C (aPKC)-interacting protein, p62, has previously been shown to interact with RIP, linking these kinases to NF-kappaB activation by tumor necrosis factor alpha (TNFalpha). The aPKCs have been implicated in the activation of IKKbeta in TNFalpha-stimulated cells and have been shown to be activated in response to interleukin-1 (IL-1). Here we demonstrate that the inhibition of the aPKCs or the down-regulation of p62 severely abrogates NF-kappaB activation by IL-1 and TRAF6, suggesting that both proteins are critical intermediaries in this pathway. Consistent with this we show that p62 selectively interacts with the TRAF domain of TRAF6 but not that of TRAF5 or TRAF2 in co-transfection experiments. The binding of endogenous p62 to TRAF6 is stimulus dependent, reinforcing the notion that this is a physiologically relevant interaction. Furthermore, we demonstrate that the N-terminal domain of TRAF6, which is required for signaling, interacts with zetaPKC in a dimerization-dependent manner. Together, these results indicate that p62 is an important intermediary not only in TNFalpha but also in IL-1 signaling to NF-kappaB through the specific adapters RIP and TRAF6.

The atypical protein kinase Cs. Functional specificity mediated by specific protein adapters.

Since its discovery more than 10 years ago, the atypical PKC (aPKC) subfamily has attracted great interest. A number of reports have shown that the kinases of this subfamily play critical roles in signaling pathways that control cell growth, differentiation and survival. Recently, several investigators have identified a number of aPKC-interacting proteins whose characterization is helping to unravel the mechanisms of action and functions of these kinases. These interactors include p62, Par-6, MEK5 and Par-4. The details of how these adapters serve to link the aPKCs to different receptor signaling pathways and substrates in response to specific stimuli are crucial not only for developing an understanding of the roles and functions of the aPKCs themselves, but also for more generally establishing a view of how specificity in signal transduction is achieved.

The downregulation of the pro-apoptotic protein Par-4 is critical for Ras-induced survival and tumor progression.

Inhibition of apoptosis is an important characteristic of oncogenic transformation. The Par-4 gene product has recently been shown to be upregulated in cells undergoing apoptotic cell death, and its ectopic expression was shown to be critical in apoptosis. We demonstrate that expression of oncogenic Ras promotes a potent reduction of Par-4 protein and mRNA levels through a MEK-dependent pathway. In addition, the expression of permanently active mutants of MEK, Raf-1 or zetaprotein kinase C but not of phosphatidylinositol 3-kinase (PI 3-kinase) is sufficient to decrease Par-4 levels. These effects are independent of p53, p16 and p19, and were detected not only in fibroblast primary cultures but also in NIH 3T3 and HeLa cells, indicating that they are not secondary to Ras actions on cell cycle regulation. Importantly, restoration of Par-4 levels to normal in Ras-transformed cells makes these cells sensitive to the pro-apoptotic actions of tumor necrosis factor-alpha under conditions in which PI 3-kinase is inhibited and also severely impairs colony formation in soft agar and tumor development in nude mice, as well as increases the sensitivity of these tumors to camptothecin. This indicates that the downregulation of Par-4 by oncogenic Ras is a critical event in tumor progression.

Insulin activates protein kinases C-zeta and C-lambda by an autophosphorylation-dependent mechanism and stimulates their translocation to GLUT4 vesicles and other membrane fractions in rat adipocytes.

In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.

Inactivation of the inhibitory kappaB protein kinase/nuclear factor kappaB pathway by Par-4 expression potentiates tumor necrosis factor alpha-induced apoptosis.

Par-4 is a novel protein identified in cells undergoing apoptosis. The ability of Par-4 to promote apoptotic cell death is dependent on the binding and inactivation of the atypical protein kinases C (PKCs). This subfamily of kinases has been reported to control nuclear factor kappaB (NF-kappaB) through the regulation of the IkappaB kinase activity. NF-kappaB activation by tumor necrosis factor alpha (TNFalpha) provides a survival signal that impairs the TNFalpha-induced apoptotic response. We show here that expression of Par-4 inhibits the TNFalpha-induced nuclear translocation of p65 as well as the kappaB-dependent promoter activity. Interestingly, Par-4 expression blocks inhibitory kappaB protein (IkappaB) kinase activity, which leads to the inhibition of IkappaB phosphorylation and degradation, in a manner that is dependent on its ability to inhibit lambda/iotaPKC. Of potential functional relevance, the expression of Par-4 allows TNFalpha to induce apoptosis in NIH-3T3 cells. In addition, the down-regulation of Par-4 levels by oncogenic Ras sensitizes cells to TNFalpha-induced NF-kappaB activation.

The interaction of p62 with RIP links the atypical PKCs to NF-kappaB activation.

The two members of the atypical protein kinase C (aPKC) subfamily of isozymes (zetaPKC and lambda/iotaPKC) are involved in the control of nuclear factor kappaB (NF-kappaB) through IKKbeta activation. Here we show that the previously described aPKC-binding protein, p62, selectively interacts with RIP but not with TRAF2 in vitro and in vivo. p62 bridges the aPKCs to RIP, whereas the aPKCs link IKKbeta to p62. In this way, a signaling cascade of interactions is established from the TNF-R1 involving TRADD/RIP/p62/aPKCs/IKKbeta. These observations define a novel pathway for the activation of NF-kappaB involving the aPKCs and p62. Consistent with this model, the expression of a dominant-negative mutant lambda/iotaPKC impairs RIP-stimulated NF-kappaB activation. In addition, the expression of either an N-terminal aPKC-binding domain of p62, or its C-terminal RIP-binding region are sufficient to block NF-kappaB activation. Furthermore, transfection of an antisense construct of p62 severely abrogates NF-kappaB activation. Together, these results demonstrate that the interaction of p62 with RIP serves to link the atypical PKCs to the activation of NF-kappaB by the TNFalpha signaling pathway.

Cleavage of zetaPKC but not lambda/iotaPKC by caspase-3 during UV-induced apoptosis.

The stimulation of caspases is a critical event in apoptotic cell death. Several kinases critically involved in cell proliferation pathways have been shown to be cleaved by caspase-mediated mechanisms. Thus, the degradation of delta protein kinase C (PKC) and MEKK-1 by caspase-3 generates activated fragments corresponding to their catalytic domains, consistent with the observations that both enzymes are important for apoptosis. In contrast, other kinases reported to have anti-apoptotic properties, such as Raf-1 and Akt, are inactivated by proteolytic degradation by the caspase system. Since the atypical PKCs have been shown to play critical roles in cell survival, in the study reported here we have addressed the potential degradation of these PKCs by the caspase system in UV-irradiated HeLa cells. Herein we show that although zetaPKC and lambda/iotaPKC are both inhibited in UV-treated cells, only zetaPKC but not lambda/iotaPKC is cleaved by a caspase-mediated process. This cleavage generates a fragment that corresponds to its catalytic domain that is enzymatically inactive. The sequence where caspase-3 cleaves zetaPKC was mapped, and a mutant resistant to degradation was shown to protect cells from apoptosis more efficiently than the wild-type enzyme.

Activation of IkappaB kinase beta by protein kinase C isoforms.

The atypical protein kinase C (PKC) isotypes (lambda/iotaPKC and zetaPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappaB by this cytokine through a mechanism that most probably involves the phosphorylation of IkappaB. The inability of these PKC isotypes to directly phosphorylate IkappaB led to the hypothesis that zetaPKC may use a putative IkappaB kinase to functionally inactivate IkappaB. Recently several groups have molecularly characterized and cloned two IkappaB kinases (IKKalpha and IKKbeta) which phosphorylate the residues in the IkappaB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-kappaB through the activation of the IKKs. We report here that alphaPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zetaPKC positively modulates IKKbeta activity but not that of IKKalpha, whereas the transfection of a zetaPKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta, which is entirely dependent on the activity of alphaPKC but not that of the atypical isoforms. In contrast, the inhibition of alphaPKC does not affect the activation of IKKbeta by TNF-alpha. Interestingly, recombinant active zetaPKC and alphaPKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha. In addition, evidence is presented here that recombinant zetaPKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappaB pathway at the level of IKKbeta activation and IkappaB degradation.

Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel (delta, epsilon) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda.

Atypical protein kinase (PK)C isoforms, zeta and lambda, have been reported to be activated by insulin via phosphoinositide 3-kinase, and have been suggested to be required for insulin-stimulated glucose transport. Here, we have examined the effects of transiently expressed wild-type (WT), constitutively active (Constit) and kinase-inactive (KI) forms of atypical PKCs, zeta and lambda, on haemagglutinin antigen (HAA)-tagged glucose transporter 4 (GLUT4) translocation in rat adipocytes, and compared these effects with each other and with those of comparable forms of conventional (alpha, beta) and novel (delta, epsilon) PKCs, which have also been proposed to be required for insulin-stimulated glucose transport. KI-PKC-zeta evoked consistent, sizeable (overall mean of 65%) inhibitory effects on insulin-stimulated, but not basal or guanosine-5'-[gamma-thio]triphosphate-stimulated, HAA-GLUT4 translocation; moreover, inhibitory effects of KI-PKC-zeta were largely reversed by co-transfection of WT-PKC-zeta. Like KI-PKC-zeta, KI-PKC-lambda inhibited insulin-stimulated HAA-GLUT4 translocation by approx. 40-60%, and the combination of KI-PKC-zeta and KI-PKC-lambda caused nearly complete (85%) inhibition. Of particular interest is the fact that inhibitory effects of KI forms of PKC-zeta and PKC-lambda were largely reversed by the opposite WT forms, i.e. PKC-lambda and PKC-zeta respectively. In contrast with KI forms of atypical PKCs, KI forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon had little or no effect on insulin-stimulated HAA-GLUT4 translocation. Concerning the question of sufficiency, overexpression of WT-PKC-zeta enhanced insulin effects on HAA-GLUT4 translocation, whereas WT forms of PKC-alpha, PKC-beta2, PKC-delta and PKC-epsilon did not affect GLUT4 translocation; furthermore, Constit PKC-zeta evoked increases in HAA-GLUT4 translocation approaching those of insulin, but Constit forms of PKC-alpha and PKC-beta2 were without effect. Our findings suggest that, among PKCs, the atypical PKCs, zeta and lambda, appear to be specifically, but interchangeably, required for insulin effects on HAA-GLUT4 translocation.

The activation of p38 and apoptosis by the inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt pathway.

Considerable attention has recently been focused on the role played by different kinase cascades in the control of apoptosis. The triggering of stress-activated kinases concomitant with the inhibition of the extracellular signal-regulated kinase (ERK) pathway has been observed in a number of cell systems undergoing programmed cell death. In addition, the activation of the phosphoinositide 3-kinase (PI 3-kinase)-Akt signaling cascade has been shown to protect from apoptosis. Here we have explored the potential role played by the inhibition of ERK in the activation of the stress kinases as well as the possible cross-talk with the PI 3-kinase pathway in HeLa cells. We show that the simple inhibition of ERK basal activity is sufficient to trigger apoptosis and p38 activation with no changes in Jun N-terminal kinase/stress-activated protein kinase. This is a process dependent on the caspases and is completely abrogated by serum. The incubation with wortmannin or the transfection of dominant negative mutants of p85 or Akt block the inhibitory function of serum, suggesting the involvement of the PI 3-kinase-Akt system. Consistent with this, expression of active mutants of PI 3-kinase and Akt inhibits p38 activation and apoptosis. We also show here that the inhibition of ERK triggers the caspase system, which is abolished by serum in a wortmannin-dependent manner. Collectively, these results demonstrate a link between ERK and the p38 apoptotic pathway that is modulated by the survival PI 3-kinase-Akt module, acting upstream the caspase system.