Martha Robles-Flores

p32 (gC1qBP) Is a General Protein Kinase C (PKC)-binding Protein INTERACTION AND CELLULAR LOCALIZATION OF P32-PKC COMPLEXES IN RAT HEPATOCYTES

The aim of this study was to identify cellular proteins that bind protein kinase C (PKC) and may influence its activity and its localization. A 32-kDa PKC-binding protein was purified to homogeneity from the Triton X-100-insoluble fraction obtained from hepatocytes homogenates. The protein was identified by NH2-terminal amino acid sequencing as the previously described mature form of p32 (gC1qR). Recombinant p32 was expressed as a glutathione S-transferase fusion protein, affinity-purified, and tested for an in vitro interaction with PKC using an overlay assay approach. All PKC isoforms expressed in rat hepatocytes interacted in vitro with p32, but the binding dependence on PKC activators was different for each one. Whereas PKCδ only binds to p32 in the presence of PKC activators, PKCζ and PKCα increase their binding when they are in the activated form. Other PKC isoforms such as β, ε, and θ bind equally well to p32 regardless of the presence of PKC activators, and PKCμ binds even better in their absence. It was also found that p32 is not a substrate for any of the PKC isoforms tested, but interestingly, its presence had a stimulatory effect (2-fold for PKCδ) on PKC activity. We also observed in vivo interaction between PKC and p32 by immunofluorescence and confocal microscopy. A time course of phorbol ester treatment of cultured rat hepatocytes (C9 cells) showed that PKCθ and p32 are constitutively associated in vivo, whereas PKCδ activation is required for its association with p32. Our data also showed that phorbol ester treatment induces a transient translocation of p32 from the cytoplasm to the cell nucleus. Together, these findings suggest that p32 may be a regulator of PKC location and function.

Hypoxia-Inducible Factors Modulate the Stemness and Malignancy of Colon Cancer Cells by Playing Opposite Roles in Canonical Wnt Signaling

This study examined the role played by hypoxia-inducible factors (HIFs) in malignant phenotype maintenance and canonical Wnt signaling. Under normoxia, we determined that both HIF-1α and HIF-2α are expressed in human colon cancer cells but not in their non-malignant counterparts. The stable knockdown of HIF-1α or HIF-2α expression induced negative effects on the malignant phenotype of colon cancer cells, with lactate production, the rate of apoptosis, migration, CXCR4-mediated chemotaxis, and tumorigenic activity all being significantly affected by HIF knockdown and with HIF-1α depletion exerting greater effects. Knockdown of these two HIF transcripts induced different and even opposite effects on β-catenin transcriptional activity in colon cancer cells with different genetic Wnt signaling pathways. In SW480 cells, HIF-2α knockdown did not affect β-catenin levels, increasing the transcriptional activity of β-catenin by inducing its nuclear accumulation, whereas HIF-1α silencing negatively affected the stability and transcriptional activity of β-catenin, inducing its exit from the nuclei and its recruitment to the cell membrane by E-cadherin. In addition, although HIF-1α depletion induced a reversal of the epithelial-to-mesenchymal transition (EMT), HIF-2α silencing altered the expression of the stem cell markers CD44, Oct4, and CD24 and of the differentiation marker CK20 in the opposite direction as HIF-1α silencing. Remarkably, HIF-2α knockdown also enhanced β-catenin transcriptional activity under hypoxia in cells that displayed normal Wnt signaling, suggesting that the gene negatively modulates canonical Wnt signaling in colon cancer cells. Taken together, our results indicate that HIFs play opposing roles in canonical Wnt signaling and are essential for the stemness and malignancy maintenance of colon cancer cells.

Protein kinase C ζ is a positive modulator of canonical Wnt signaling pathway in tumoral colon cell lines

The colonic epithelium is a continuously renewing tissue with a dynamic turnover of cells. Wnt pathway is a key regulator of its homeostasis and is altered in a large proportion of colon cancers. Protein kinase C (PKC) family of serine/threonine kinases are also involved in colon tumor formation and progression; however, the molecular role played by them in the Wnt pathway, is poorly understood. Reciprocal coimmunoprecipitation and immunofluorescence studies revealed that PKCζ interacts with β-catenin mainly in tumoral colon cells, which overexpressed this PKC isoform. The pharmacological inhibition, the small interference RNA-mediated knockdown of PKCζ or the expression of a dominant-negative form of it in tumoral SW480 cells, blocked in a dose-dependent manner the constitutive transcriptional activity mediated by β-catenin, the cell proliferation and the expression of the Wnt target gene c-myc. Remarkably, the PKCζ stably depleted cells exhibited diminished tumorigenic activity in grafted mice. We show that PKCζ functions in a mechanism that does not involve β-catenin degradation since the effects produced by PKCζ inhibition were also obtained in the presence of proteosome inhibitor and in cells expressing a β-catenin degradation-resistant mutant. It was found that PKCζ activity regulates the nuclear localization of β-catenin since PKCζ inhibition induces a rapid export of β-catenin from the nucleus to the cytoplasm in a Leptomycin B sensitive manner. Taken together, our results indicate that the atypical PKCζ plays an important role in the positive regulation of canonical Wnt pathway.

Posttranslational modifications on protein kinase c isozymes. Effects of epinephrine and phorbol esters

The posttranslational modifications induced on PKC isozymes as result of their activation were investigated. Reciprocal immunoprecipitations followed by Western blot analysis demonstrated that all PKC isozymes expressed in rat hepatocytes are modified by tyrosine nitration and tyrosine phosphorylation in different ways upon exposure of cells to a direct PKC activator (TPA), or to an extracellular ligand known to activate PKC-dependent pathways (epinephrine). Our data demonstrate for the first time that all PKC isozymes are also dynamically modified by O-linked beta-N-acetylglucosamine (O-GlcNAc); the presence of this modification was confirmed in part by FT-ICR mass spectrometry analysis. Interestingly, the O-GlcNAc modified Ser or Thr were mapped at similar positions in several PKC isozymes. The biochemical meaning of these posttranslational modifications was investigated for PKC alpha and delta. It was found that the PKC phosphorylation status of both isozymes in tyrosine and serine residues seems to regulate directly the enzyme activity since catalytic inactivation correlate with dephosphorylation of Ser at the C-terminus autophosphorylation sites of each PKC isozyme, and with an increase in the level of tyrosine phosphorylation. Whereas none of the other posttranslational modifications showed per se a direct effect in PKC delta activity, increased tyrosine nitration and O-GlcNAc modifications correlate negatively with PKCalpha activity.

Protein kinase C in Wnt signaling: Implications in cancer initiation and progression

Although it is well known that Wnt and protein kinase C (PKC) signaling pathways are both involved in carcinogenesis and tumor progression, their synergistic contribution to these processes or the crosstalk between them has just recently been approached. The Wnt and PKC signaling are involved in many cellular functions including proliferation, differentiation, survival, apoptosis, cytoskeletal remodeling, and cell motility. Canonical Wnt signaling has been well characterized as one of the most important contributors to tumorigenesis, and it has been implicated in many types of solid tumors. PKC is one of the key targets of noncanonical Wnt signaling, particularly in the Wnt/Ca2+ pathway. Recently, data have implicated components of noncanonical Wnt/Ca2+ and Wnt/planar cell polarity signaling in directly promoting the invasiveness and malignant progression of diverse forms of human cancer. But, unlike the canonical pathway, defining the roles of noncanonical Wnt signaling in human cancer is in its infancy. In this review, we provide a concise description of the current knowledge of the interaction between PKC and Wnt pathways and discuss the role of this crosstalk in cancer initiation and progression.

Differences in phorbol ester-induced decrease of the activity of protein kinase C isozymes in rat hepatocytes

Two main forms of protein kinase C (PKC) activity were found in rat hepatocytes using DEAE-cellulose chromatography: PKC 1 and PKC 2. Treatment of cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 15 min caused a marked loss of PKC 1 activity and only a small loss of PKC 2 activity. Hydroxyapatite column chromatography resolved PKC 1 into three distinct peaks 1a, 1b and 1c, and PKC 2 into four peaks 2a, 2b, 2c and 2d. Immunoblot analysis with isozyme-specific monoclonal antibodies identified peak 1a as PKC-beta and peak 1b as PKC-alpha; the other peaks of activity were not identified. Treatment with TPA provoked a loss of activity of peaks 1b (PKC-alpha) and 1c, whereas peak 1a (PKC-beta) activity was not affected. The peaks of activity corresponding to PCK 2 did not show any major change due to TPA treatment except peak 2d that decreased. The apparent disappearance of PKC histone-kinase activity induced by TPA was also observed using other substrates (protamine or vinculin). The TPA-induced decrease in activity occurs in a time-dependent and dose-dependent fashion. However, the time-courses, the extent of depletion and the potency order of phorbol esters in induction of an activity decrease in the two groups of isoforms exhibited substantial differences.

Protein Kinase C Delta Negatively Modulates Canonical Wnt Pathway and Cell Proliferation in Colon Tumor Cell Lines

The tumor suppressor Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers. Alterations in Protein kinase C (PKC) isozyme expression and aberrant regulation also comprise early events in intestinal carcinomas. Here we show that PKCδ expression levels are decreased in colon tumor cell lines with respect to non-malignant cells. Reciprocal co-immunoprecipitation and immunofluorescence studies revealed that PKCδ interacts specifically with both full-length (from non-malignant cells) and truncated APC protein (from cancerous cells) at the cytoplasm and at the cell nucleus. Selective inhibition of PKCδ in cancer SW480 cells, which do not possess a functional β-catenin destruction complex, did not affect β-catenin-mediated transcriptional activity. However, in human colon carcinoma RKO cells, which have a normal β-catenin destruction complex, negatively affected β-catenin-mediated transcriptional activity, cell proliferation, and the expression of Wnt target genes C-MYC and CYCLIN D1. These negative effects were confirmed by siRNA-mediated knockdown of PKCδ and by the expression of a dominant negative form of PKCδ in RKO cells. Remarkably, the PKCδ stably depleted cells exhibited augmented tumorigenic activity in grafted mice. We show that PKCδ functions in a mechanism that involves regulation of β-catenin degradation, because PKCδ inhibition induces β-catenin stabilization at the cytoplasm and its nuclear presence at the C-MYC enhancer even without Wnt3a stimulation. In addition, expression of a dominant form of PKCδ diminished APC phosphorylation in intact cells, suggesting that PKCδ may modulate canonical Wnt activation negatively through APC phosphorylation.

Protein kinase C isoforms from Giardia duodenalis : identification and functional characterization of a β-like molecule during encystment

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate many different cellular processes such as cell growth and differentiation in eukaryotic cells. Using specific polyclonal antibodies raised against mammalian PKC isoforms, it was demonstrated here for the first time that Giardia duodenalis expresses several PKC isoforms (beta, delta, epsilon, theta and zeta). All PKC isoforms detected showed changes in their expression pattern during encystment induction. In addition, selective PKC inhibitors blocked the encystment in a dose-dependent manner, suggesting that PKC isozymes may play important roles during this differentiation process. We have characterized here the only conventional-type PKC member found so far in Giardia, which showed an increased expression and changes in its intracellular localization pattern during cyst formation. The purified protein obtained by chromatography on DEAE-cellulose followed by size-exclusion chromatography, displayed in vitro kinase activity using histone HI-IIIS as substrate, which was dependent on cofactors required by conventional PKCs, i.e., phospholipids and calcium. An open reading frame in the Giardia Genome Database that encodes a homolog of PKCβ catalytic domain was identified and cloned. The expressed recombinant protein was also recognized by a mammalian anti-PKCβ antibody and was referred as giardial PKCβ on the basis of all these experimental evidence.

Glycogen synthase kinase 3 in Wnt signaling pathway and cancer

Glycogen synthase kinase 3 (GSK‐3) was first discovered in 1980 as one of the key enzymes of glycogen metabolism. Since then, GSK‐3 has been revealed as one of the master regulators of a diverse range of signaling pathways, including those activated by Wnts, participating in the regulation of numerous cellular functions, suggesting that its activity is tightly regulated. Numerous studies have pointed to an association of GSK‐3 dysregulation with the onset and progression of human diseases, including diabetes mellitus, obesity, inflammation, neurological illnesses, and cancer. Therefore, GSK‐3 is recognized as an attractive therapeutic target in multiple disorders. However, the great number of substrates that are phosphorylated by GSK‐3 has raised the question of whether this limits its feasibility as a therapeutic target because of the potential disruption of many cellular processes and also by the fear that inhibition of GSK‐3 may stimulate or aid in malignant transformation, as GSK‐3 can phosphorylate pro‐oncogenic factors. This mini review focuses on the role played by GSK‐3 in Wnt signaling pathway and cancer using as model colon cancer.

Phosphorylation of Zona Occludens-2 by Protein Kinase Cε Regulates Its Nuclear Exportation

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCepsilon. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.