A major 62-kD intranuclear matrix polypeptide is a component of metaphase chromosomes.

We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.

Overexpression of protein kinase C betaII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis.

Protein kinase C betaII (PKC betaII) has been implicated in proliferation of the intestinal epithelium. To investigate PKC betaII function in vivo, we generated transgenic mice that overexpress PKC betaII in the intestinal epithelium. Transgenic PKC betaII mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC betaII mice exhibit elevated colonic beta-catenin levels and decreased glycogen synthase kinase 3beta activity, indicating that PKC betaII stimulates the Wnt/adenomatous polyposis coli (APC)/beta-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC betaII in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta-catenin signaling pathway.

Evi1 is a survival factor which conveys resistance to both TGFbeta- and taxol-mediated cell death via PI3K/AKT.

In hematopoietic cells the transforming potential of the ecotropic viral integration site 1 (Evi1) oncogene is thought to be dependent upon the ability to inhibit TGFbeta signaling. Although Evi1 has recently been implicated in certain epithelial cancers, the effects of Evi1 on transformation and TGFbeta signaling in epithelial cells are not completely understood. Herein, we have determined the effects of Evi1 on TGFbeta signaling in intestinal epithelial cells. Stable expression of Evi1 in non-transformed intestinal epithelial cells inhibited induction of some Smad3-dependent TGFbeta target genes, such as PAI1. However, TGFbeta-mediated induction of cellular adhesion signaling components such as integrin1 and paxillin was not inhibited by Evi1; nor did Evi1 inhibit TGFbeta-mediated epithelial to mesenchymal transition. Likewise, Evi1 did not inhibit TGFbeta-mediated downregulation of cyclin D1 or block TGFbeta-mediated growth inhibition. However, Evi1 did inhibit TGFbeta-mediated apoptosis by a process that involves phosphoinositide-3-kinase (PI3K) and its downstream effector AKT. The ability of Evi1 to suppress apoptosis is not restricted to TGFbeta-mediated cell death, since Evi1 also protects intestinal epithelial cells from taxol-mediated apoptosis. Evi1 is overexpressed in some human colon cancer cell lines, and overexpression is associated with amplification of the Evi1 gene. Knockdown of Evi1 by siRNA inhibited AKT phosphorylation in HT-29 human colon cancer cells and increased their sensitivity to taxol-mediated apoptosis. These data indicate that Evi1 functions as a survival gene in intestinal epithelial cells and colon cancer cells, activating PI3K/AKT and conveying resistance to both physiological and therapeutic apoptotic stimuli.

PKCι regulates nuclear YAP1 localization and ovarian cancer tumorigenesis.

Atypical protein kinase Cι (PKCι) is an oncogene in lung and ovarian cancer. The PKCι gene PRKCI is targeted for frequent tumor-specific copy number gain (CNG) in both lung squamous cell carcinoma (LSCC) and ovarian serous carcinoma (OSC). We recently demonstrated that in LSCC cells PRKCI CNG functions to drive transformed growth and tumorigenicity by activating PKCι-dependent cell autonomous Hedgehog (Hh) signaling. Here, we assessed whether OSC cells harboring PRKCI CNG exhibit similar PKCι-dependent Hh signaling. Surprisingly, we find that whereas PKCι is required for the transformed growth of OSC cells harboring PRKCI CNG, these cells do not exhibit PKCι-dependent Hh signaling or Hh-dependent proliferation. Rather, transformed growth of OSC cells is regulated by PKCι-dependent nuclear localization of the oncogenic transcription factor, YAP1. Lentiviral shRNA-mediated knockdown (KD) of PKCι leads to decreased nuclear YAP1 and increased YAP1 binding to angiomotin (AMOT), which sequesters YAP1 in the cytoplasm. Biochemical analysis reveals that PKCι directly phosphorylates AMOT at a unique site, Thr750, whose phosphorylation inhibits YAP1 binding. Pharmacologic inhibition of PKCι decreases YAP1 nuclear localization and blocks OSC tumor growth in vitro and in vivo. Immunohistochemical analysis reveals a strong positive correlation between tumor PKCι expression and nuclear YAP1 in primary OSC tumor samples, indicating the clinical relevance of PKCι-YAP1 signaling. Our results uncover a novel PKCι-AMOT-YAP1 signaling axis that promotes OSC tumor growth, and provide a rationale for therapeutic targeting of this pathway for treatment of OSC.

Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis.

Protein kinase C (PKC) has been implicated in colon carcinogenesis in humans and in rodent models. However, little is known about the specific role of individual PKC isozymes in this process. We recently demonstrated that elevated expression of PKC betaII in the colonic epithelium induces hyperproliferation in vivo (N. R. Murray et al., J. Cell Biol., 145: 699-711, 1999). Because hyperproliferation is a major risk factor for colon cancer, we assessed whether specific alterations in PKC betaII expression occur during azoxymethane-induced colon carcinogenesis in mice. An increase in PKC betaII expression was observed in preneoplastic lesions (aberrant crypt foci, 3.7-fold) compared with saline-treated animals, and in colon tumors (7.8-fold; P = 0.011) compared with uninvolved colonic epithelium. In contrast, PKC alpha and PKC betaI (a splicing variant of PKC betaII) expression was slightly decreased in aberrant crypt foci and dramatically reduced in colon tumors. Quantitative reverse transcription-PCR analysis revealed that PKC mRNA levels do not directly correlate with PKC protein levels, indicating that PKC isozyme expression is likely regulated at the posttranscriptional/translational level. Finally, transgenic mice expressing elevated PKC betaII in the colonic epithelium exhibit a trend toward increased colon tumor formation after exposure to azoxymethane. Taken together, our results demonstrate that elevated expression of PKC betaII is an important early, promotive event that plays a role in colon cancer development.

NF-kappaB/RelA transactivation is required for atypical protein kinase C iota-mediated cell survival.

In chronic myelogenous leukemia (CML), the oncogene bcr-abl encodes a dysregulated tyrosine kinase that inhibits apoptosis. We showed previously that human erythroleukemia K562 cells are resistant to antineoplastic drug (taxol)-induced apoptosis through the atypical protein kinase C iota isozyme (PKC iota), a kinase downstream of Bcr-Abl. The mechanism(s) by which PKC iota mediates cell survival to taxol is unknown. Here we demonstrate that PKC iota requires the transcription factor nuclear factor-kappaB (NF-kappaB) to confer cell survival. At apoptosis-inducing concentrations, taxol weakly induces IkappaB(alpha) proteolysis and NF-kappaB translocation in K562 cells, but potently induces its transcriptional activity. Inhibition of NF-kappaB activity (by blocking IkappaB(alpha) degradation) significantly sensitizes cells to taxol-induced apoptosis. Likewise, K562 cells expressing antisense PKC iota mRNA or kinase dead PKC iota (PKC iota-KD) are sensitized to taxol; these cells are rescued from apoptosis by NF-kappaB overexpression. Expression of constitutively active PKC iota (PKC iota-CA) upregulates NF-kappaB transactivation and rescues cells from apoptosis in the absence of Bcr-Abl tyrosine kinase activity. Using a chimeric GAL4-RelA transactivator, we find that taxol potently activates GAL4-RelA-dependent transcription. This activation was further upregulated by expression of PKC iota-CA and inhibited by expression of PKC iota-KD. Our results indicate that RelA transactivation is an important downstream target of the PKC iota-mediated Bcr-Abl signaling pathway and is required for resistance to taxol-induced apoptosis.

Pituitary expression of protein kinase C isotypes during early development.

Protein kinase C (PKC) is a critical regulator of signal transduction and cell function in many tissues, including pituitary. Although PKC influences pituitary hormone secretion in adults, its role in determining characteristic perinatal patterns of hormone secretion and synthesis is not known, and the expression of major PKC isotypes in perinatal pituitary is poorly defined. We therefore determined the developmental, cell-specific expression of the major PKC isotypes, using Western analysis and double label immunohistochemistry, in pituitaries of perinatal and mature rats. Expression of specific PKC isotypes was strikingly age-dependent. Pituitary expression of PKC alpha was particularly high in neonates and declined significantly with age, with levels in adult rats approximately half those of neonates as assessed by Western analysis. Similarly, immunohistochemistry indicated that PKC alpha was less abundant in adult than in neonatal pituitaries; the most intensely staining cells of both age groups were identified as somatotrophs and gonadotrophs. In contrast to PKC alpha, pituitary expression of PKC epsilon increased approximately two-fold with advancing age as assessed by Western analysis; this age-dependent pattern was confirmed by immunohistochemistry. Perinatal pituitaries expressed PKC epsilon in some somatotrophs and in all gonadotrophs, whereas PKC epsilon expression was limited to gonadotrophs in the mature pituitary. Pituitary expression of PKC betaII, delta, and zeta did not differ with age, and PKC gamma was not detected in pituitaries of any age group. These results indicate that expression of PKC isotypes within the pituitary is developmentally regulated in a cell-specific and isotype-specific manner, and are consistent with the concept that PKC contributes to the regulation of pituitary function during early development.

Protein kinase Cα suppresses Kras-mediated lung tumor formation through activation of a p38 MAPK-TGFß signaling axis.

Protein kinase C alpha (PKCα) can activate both pro- and anti-tumorigenic signaling depending upon cellular context. Here, we investigated the role of PKCα in lung tumorigenesis in vivo. Gene expression data sets revealed that primary human non-small lung cancers (NSCLC) express significantly decreased PKCα levels, indicating that loss of PKCα expression is a recurrent event in NSCLC. We evaluated the functional relevance of PKCα loss during lung tumorigenesis in three murine lung adenocarcinoma models (LSL-Kras, LA2-Kras and urethane exposure). Genetic deletion of PKCα resulted in a significant increase in lung tumor number, size, burden and grade, bypass of oncogene-induced senescence, progression from adenoma to carcinoma and a significant decrease in survival in vivo. The tumor promoting effect of PKCα loss was reflected in enhanced Kras-mediated expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells, both in vitro and in vivo. LSL-Kras/Prkca(-/-) mice exhibited a decrease in phospho-p38 MAPK in BASCs in vitro and in tumors in vivo, and treatment of LSL-Kras BASCs with a p38 inhibitor resulted in increased colony size indistinguishable from that observed in LSL-Kras/Prkca(-/-) BASCs. In addition, LSL-Kras/Prkca(-/-) BASCs exhibited a modest but reproducible increase in TGFß1 mRNA, and addition of exogenous TGFß1 to LSL-Kras BASCs results in enhanced growth similar to untreated BASCs from LSL-Kras/Prkca(-/-) mice. Conversely, a TGFßR1 inhibitor reversed the effects of PKCα loss in LSL-Kras/Prkca(-/-) BASCs. Finally, we identified the inhibitors of DNA binding (Id) Id1-3 and the Wilm's Tumor 1 as potential downstream targets of PKCα-dependent tumor suppressor activity in vitro and in vivo. We conclude that PKCα suppresses tumor initiation and progression, at least in part, through a PKCα-p38MAPK-TGFß signaling axis that regulates tumor cell proliferation and Kras-induced senescence. Our results provide the first direct evidence that PKCα exhibits tumor suppressor activity in the lung in vivo.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

Ect2 links the PKCiota-Par6alpha complex to Rac1 activation and cellular transformation.

Protein kinase Ciota (PKCiota) promotes non-small cell lung cancer (NSCLC) by binding to Par6alpha and activating a Rac1-Pak-Mek1,2-Erk1,2 signaling cascade. The mechanism by which the PKCiota-Par6alpha complex regulates Rac1 is unknown. Here we show that epithelial cell transforming sequence 2 (Ect2), a guanine nucleotide exchange factor for Rho family GTPases, is coordinately amplified and overexpressed with PKCiota in NSCLC tumors. RNA interference-mediated knockdown of Ect2 inhibits Rac1 activity and blocks transformed growth, invasion and tumorigenicity of NSCLC cells. Expression of constitutively active Rac1 (RacV12) restores transformation to Ect2-deficient cells. Interestingly, the role of Ect2 in transformation is distinct from its well-established role in cytokinesis. In NSCLC cells, Ect2 is mislocalized to the cytoplasm where it binds the PKCiota-Par6alpha complex. RNA interference-mediated knockdown of either PKCiota or Par6alpha causes Ect2 to redistribute to the nucleus, indicating that the PKCiota-Par6alpha complex regulates the cytoplasmic localization of Ect2. Our data indicate that Ect2 and PKCiota are genetically and functionally linked in NSCLC, acting to coordinately drive tumor cell proliferation and invasion through formation of an oncogenic PKCiota-Par6alpha-Ect2 complex.

Matrix metalloproteinase-10 is a critical effector of protein kinase Ciota-Par6alpha-mediated lung cancer.

Protein kinase Ciota (PKCiota) drives transformed growth of non-small cell lung cancer (NSCLC) cells through the Rho family GTPase Rac1. We show here that PKCiota activates Rac1 in NSCLC cells by formation of a PKCiota-Par6alpha complex that drives anchorage-independent growth and invasion through activation of matrix metalloproteinase-10 (MMP-10) expression. RNAi-mediated knockdown of PKCiota, Par6alpha or Rac1 expression inhibits NSCLC transformation and MMP-10 expression in vitro. Expression of wild-type Par6alpha in Par6alpha-deficient cells restores transformation and MMP-10 expression, whereas expression of Par6alpha mutants that either cannot bind PKCiota (Par6alpha-K19A) or couple to Rac1 (Par6alpha-DeltaCRIB) do not. Knockdown of MMP-10 expression blocks anchorage-independent growth and invasion of NSCLC cells and addition of catalytically active MMP-10 to PKCiota- or Par6alpha-deficient cells restores anchorage-independent growth and invasion. Dominant-negative PKCiota inhibits tumorigenicity and MMP-10 expression in subcutaneous NSCLC tumors. MMP-10 and PKCiota are coordinately overexpressed in primary NSCLC tumors, and tumor MMP-10 expression predicts poor survival in NSCLC patients. Our data define a PKCiota-Par6alpha-Rac1 signaling axis that drives anchorage-independent growth and invasion of NSCLC cells through induction of MMP-10 expression.

Targeting the oncogenic protein kinase Ciota signalling pathway for the treatment of cancer.

PKC (protein kinase C) isoenzymes are key signalling components involved in the regulation of normal cell proliferation, differentiation, polarity and survival. The aberrant regulation of PKC isoenzymes has been implicated in the development of many human diseases including cancer [Fields and Gustafson (2003) Methods Mol. Biol. 233, 519-537]. To date, however, only one PKC isoenzyme, the aPKC [atypical PKCiota (protein kinase Ciota)], has been identified as a human oncogene [Regala, Weems, Jamieson, Khoor, Edell, Lohse and Fields (2005) Cancer Res. 65, 8905-8911]. PKCiota has also proven to be a useful prognostic marker and legitimate target for the development of novel pharmacological agents for the treatment of cancer. The PKCiota gene resides at chromosome 3q26 and is a frequent target of tumour-specific gene amplification in multiple forms of human cancer. PKCiota gene amplification in turn drives PKCiota overexpression in these cancers. Genetic disruption of PKCiota expression blocks multiple aspects of the transformed phenotype of human cancer cells including transformed growth in soft agar, invasion through Matrigel and growth of subcutaneous tumours in nude mice. Genetic dissection of oncogenic PKCiota signalling mechanisms demonstrates that PKCiota drives transformed growth by activating a PKCiota --> Rac1 --> PAK (p21-activated kinase) --> MEK [MAPK (mitogen-activated protein kinase) 1,2/ERK (extracellular-signal-regulated kinase) kinase] 1,2 signalling pathway [Regala, Weems, Jamieson, Copland, Thompson and Fields (2005) J. Biol. Chem. 280, 31109-31115]. The transforming activity of PKCiota requires the N-terminal PB1 (Phox-Bem1) domain of PKCiota, which serves to couple PKCiota with downstream effector molecules. Hence, there exists a strong rationale for developing novel cancer therapeutics that target the PB1 domain of PKCiota and thereby disrupt its interactions with effector molecules. Using a novel high-throughput drug screen, we identified compounds that can disrupt PB1-PB1 domain interactions between PKCiota and the adaptor molecule Par6 [Stallings-Mann, Jamieson, Regala, Weems, Murray and Fields (2006) Cancer Res. 66, 1767-1774]. Our screen identified the gold compounds ATG (aurothioglucose) and ATM (aurothiomalate) as specific inhibitors of the PB1-PB1 domain interaction between PKCiota and Par6 that exhibit anti-tumour activity against NSCLC (non-small-cell lung cancer) both in vitro and in vivo. Structural analysis, site-directed mutagenesis and modelling indicate that ATM specifically targets the PB1 domain of PKCiota to mediate its anti-tumour activity [Erdogan, Lamark, Stallings-Mann, Lee, Pellechia, Thompson, Johansen and Fields (2006) J. Biol. Chem. 281, 28450-28459]. Taken together, our recent work demonstrates that PKCiota signalling is required for transformed growth of human tumours and is an attractive target for development of mechanism-based cancer therapies. ATM is currently in Phase I clinical trials for the treatment of NSCLC.

Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells.

The promyelocytic leukemia (HL60) cell line differentiates into monocyte-like cells after treatment with phorbol dibutyrate (PBt2). In contrast, bryostatin 1 (bryo), a structurally distinct protein kinase C (PKC) activator, does not induce differentiation and blocks the cytostatic effect of PBt2. The divergent responses to these agents correlate with activation of a PKC-like activity at the nucleus in response to bryo but not PBt2 (Fields, A. P., Pettit, G. R., and May, W.S. (1988) J. Biol. Chem. 263, 8253-8260). In the present study, this nuclear PKC-like activity (termed PKCn) was isolated from HL60 cells and shown to phosphorylate its known nuclear substrate, lamin B. PKCn-mediated phosphorylation of nuclear envelope-associated lamin B in vitro is calcium-dependent and is stimulated by bryo and 1,2-dioctanoylglycerol (DiC8), but not PBt2. In contrast, PKCn-mediated phosphorylation of histone IIIS is stimulated equally by all three activators. PKCn mediates calcium- and phosphatidylserine-dependent phosphorylation of both histone IIIS and partially purified lamin B. PKCn activity can be inhibited by an anti-PKC monoclonal antibody which specifically inhibits PKC. Isotype-specific PKC antibodies identify PKCn as beta II-PKC. Immunoblot analysis indicates that HL60 cells express both alpha- and beta II-PKC but no beta I- or gamma-PKC. Treatment of intact cells with bryo for 30 min leads to complete translocation of both alpha- and beta II-PKC from the cytosol to the membrane fractions. Approximately 8-10% of the total beta II-PKC (and less than 0.3% of the alpha-PKC) is found associated with the nuclear membrane of bryo-treated cells. In contrast, PBt2 treatment leads to complete translocation of alpha-PKC, but only partial translocation of beta II-PKC to the plasma membrane fraction. Neither PKC isotype is found associated with the nuclear membrane of PBt2-treated cells. These data demonstrate that alpha- and beta II-PKC differ with respect to activator responsiveness, intracellular distribution, and substrate specificity and indicate that their selective activation at distinct intracellular sites, including the nucleus, can have a dramatic effect on resulting cellular responses.

The regulation of mitotic nuclear envelope breakdown: a role for multiple lamin kinases.

The chapter reviews the structure and function of the nuclear envelope and describes its dynamic structural changes during cell cycle. Particular emphasis is placed on the regulation of mitotic nuclear envelope breakdown (NEBD), the process by which the physical barrier between cytoplasm and nucleus is dissolved to allow for cell division. The literature suggesting the involvement of multiple protein kinases in NEBD is reviewed and evidence is presented that multiple mitotic lamin kinases, including p34cdc2/cyclin B kinase and protein kinase C, play key roles in mitotic nuclear lamina disassembly. Finally, a model for regulation of mitotic nuclear lamina disassembly by multi-site phosphorylation is described.

Phosphatidylglycerol is a physiologic activator of nuclear protein kinase C.

A major mechanism by which protein kinase C (PKC) function is regulated is through the selective targeting and activation of individual PKC isotypes at distinct subcellular locations. PKC betaII is selectively activated at the nucleus during G2 phase of cell cycle where it is required for entry into mitosis. Selective nuclear activation of PKC betaII is conferred by molecular determinants within the carboxyl-terminal catalytic domain of the kinase (Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9156-9160). We previously described a lipid-like PKC activator in nuclear membranes, termed nuclear membrane activation factor (NMAF), that potently stimulates PKC betaII activity through interactions involving this domain (Murray, N. R., Burns, D. J., and Fields, A. P. (1994) J. Biol. Chem. 269, 21385-21390). We have now identified NMAF as phosphatidylglycerol (PG), based on several lines of evidence. First, NMAF cofractionates with PG as a single peak of activity through multiple chromatographic separations and exhibits phospholipase sensitivity identical to that of PG. Second, purified PG, but not other phospholipids, exhibits dose-dependent NMAF activity. Third, defined molecular species of PG exhibit different abilities to stimulate PKC betaII activity. 1,2-Dioleoyl-PG possesses significantly higher activity than other PG species, suggesting that both fatty acid side chain composition and the glycerol head group are important determinants for activity. Fourth, in vitro binding studies demonstrate that PG binds to the carboxyl-terminal region of PKC betaII, the same region we previously implicated in NMAF-mediated activation of PKC betaII. Taken together, our results indicate that specific molecular species of nuclear PG function to physiologically regulate PKC betaII activity at the nucleus.

Mapping of a molecular determinant for protein kinase C betaII isozyme function.

In human erythroleukemia (K562) cells, the highly related protein kinase C (PKC) alpha and PKC betaII isozymes serve distinct functions in cellular differentiation and proliferation, respectively. Previous studies using two domain switch PKC chimera revealed that the catalytic domains of PKC alpha and betaII contain molecular determinants important for isozyme-specific function (Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9156-9160). We have now analyzed a panel of PKC chimeras to determine the specific region within the catalytic domain important for PKC betaII function. A cellular assay for PKC betaII function was devised based on the finding that PKC betaII selectively translocates to the nucleus and phosphorylates nuclear lamin B in response to the PKC activator bryostatin. This response is strictly dependent upon expression of PKC betaII or a PKC chimera that functions like PKC betaII. We demonstrate that a PKC alpha/betaII chimera containing only the carboxyl-terminal 13 amino acids from PKC betaII (betaII V5) is capable of nuclear translocation and lamin B phosphorylation. These results are consistent with our recent observation that the PKC betaII V5 region binds to phosphatidylglycerol (PG), a potent and selective PKC betaII activator present in the nuclear membrane (Murray, N. R., and Fields, A. P. (1998) J. Biol. Chem. 273, 11514-11520). Soluble betaII V5 peptide selectively inhibits PG-stimulated PKC betaII activity in a dose-dependent fashion, indicating that PG-mediated activation of PKC betaII involves interactions with the betaII V5 region of the enzyme. We conclude that betaII V5 is a major determinant for PKC betaII nuclear function and suggest a model in which binding of PG to the betaII V5 region stimulates nuclear PKC betaII activity during G2 phase of the cell cycle.

Atypical protein kinase C iota protects human leukemia cells against drug-induced apoptosis.

Protein kinase C (PKC) isozymes play distinct roles in cellular function. In human K562 leukemia cells, PKC alpha is important for cellular differentiation and PKC betaII is required for proliferation. In this report, we assess the role of the atypical PKC isoform PKC iota in K562 leukemia cell physiology. K562 cells were stably transfected with expression plasmids containing the cDNA for human PKC iota in sense or antisense orientation to increase or decrease cellular PKC iota levels, respectively. Overexpression or inhibition of expression of PKC iota had no significant effect on the proliferative capacity of K562 cells nor their sensitivity to phorbol myristate acetate-induced cytostasis and megakaryocytic differentiation, suggesting that PKC iota does not play a critical role in these processes. Rather, PKC iota serves to protect K562 cells against drug-induced apoptosis. K562 cells, which are resistant to most apoptotic agents, undergo apoptosis when treated with the protein phosphatase inhibitor okadaic acid (OA). Overexpression of PKC iota leads to increased resistance to OA-induced apoptosis whereas inhibition of PKC iota expression sensitizes cells to OA-induced apoptosis. Overexpression of the related atypical PKC zeta has no protective effect, demonstrating that the effect is isotype-specific. PKC iota also protects K562 cells against taxol-induced apoptosis, indicating that it plays a general protective role against apoptotic stimuli. These data support a role for PKC iota in leukemia cell survival.

betaII protein kinase C is required for the G2/M phase transition of cell cycle.

Entry into mitosis requires the coordinated action of multiple mitotic protein kinases. In this report, we investigate the involvement of protein kinase C in the control of mitosis in human cells. Treatment of synchronized HL60 cells with the highly selective protein kinase C (PKC) inhibitor chelerythrine chloride leads to profound cell cycle arrest in G2 phase. The cellular effects of chelerythrine are not due to either direct or indirect inhibition of the known mitotic regulator p34(cdc2)/cyclin B kinase. Rather, several lines of evidence demonstrate that chelerythrine-mediated G2 phase arrest results from selective inhibition and degradation of betaII protein kinase C. First, chelerythrine causes dose-dependent inhibition of betaII PKC in vitro with an IC50 identical to that for G2 phase blockade in whole cells. Second, chelerythrine specifically inhibits betaII PKC-mediated lamin B phosphorylation and mitotic nuclear lamina disassembly. Third, chelerythrine leads to selective loss of betaII PKC during G2 phase in synchronized cells. Fourth, chelerythrine mediates activation-dependent degradation of PKC, indicating that betaII PKC is selectively activated during G2 phase of cell cycle. Taken together, these data demonstrate that betaII PKC activation at G2 phase is required for mitotic nuclear lamina disassembly and entry into mitosis and that betaII PKC-mediated phosphorylation of nuclear lamin B is important in these events.

Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds.

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.

Activation of transcription factor SAF involves its phosphorylation by protein kinase C.

The transcription factor serum amyloid A (SAA)-activating factor (SAF), a family of zinc finger proteins, plays a significant role in the induced expression of the SAA gene. Activity of SAF is regulated by a phosphorylation event involving serine/threonine protein kinase (Ray, A., Schatten, H., and Ray, B. K. (1999) J. Biol. Chem. 274, 4300-4308; Ray, A., and Ray, B. K. (1998) Mol. Cell. Biol. 18, 7327-7335). However, the identity of the protein kinase has so far remained unknown. Induction of SAA by phorbol 12-myristate 13-acetate, a known agonist of protein kinase C (PKC), suggested a potential role of the PKC signaling pathway in the activation process. The DNA binding activity of endogenous SAF was increased by agonists of PKC. In vitro phosphorylation of SAF-1 by PKC-beta markedly increased its DNA binding ability. Consistent with these findings, treatment of cells with activators of PKC or overexpression of PKC-betaII in transfected cells increased expression of an SAF-regulated promoter. Further analysis with a GAL4 reporter system indicated that PKC-mediated phosphorylation mostly increases the DNA binding activity of SAF-1. Together these data indicated that the PKC signaling pathway plays a major role in controlling expression of SAF-regulated genes by increasing the interaction between promoter DNA and phosphorylated SAF.