Sequential posttranslational modifications regulate PKC degradation

Cross-talk among different types of posttranslational modifications (PTMs) has emerged as an important regulatory mechanism for protein function. Here we elucidate a mechanism that controls PKCα stability via a sequential cascade of PTMs. We demonstrate that PKCα dephosphorylation decreases its sumoylation, which in turn promotes its ubiquitination and ultimately enhances its degradation via the ubiquitin-proteasome pathway. These findings provide a molecular explanation for the activation-induced down-regulation of PKC proteins.     

Hsp70 Interacts with the Retroviral Restriction Factor TRIM5�� and Assists the Folding of TRIM5��

Tripartite motif (TRIM) protein TRIM5α has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5α is not known. In this study, we showed that transiently overexpressed TRIM5α causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5α suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5α-binding partner. The interaction between Hsp70 and TRIM5α was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5α-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5α, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5α-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5α. Studies using a series of TRIM5α deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5α structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5α, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5α in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5α-induced cellular insults.     

CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70

Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1(G93A) transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome.     

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.     

A hypoxia-induced decrease of either MICA/B or Hsp70 on the membrane of tumor cells mediates immune escape from NK cells

Recent findings suggest that hypoxia of the tumor microenvironment contributes to immune escape from natural killer (NK) cell-mediated cytotoxicity. Heat shock protein 70 (Hsp70) and the stress-regulated major histocompatibility class I chain-related protein A and B (MICA/B) both serve as ligands for activated NK cells when expressed on the cell surface of tumor cells. Herein, we studied the effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) on the membrane expression of these NK cell ligands in H1339 with high and MDA-MB-231 tumor cells with low basal HIF-1α levels and its consequences on NK cell-mediated cytotoxicity. We could show that a hypoxia-induced decrease in the membrane expression of MICA/B and Hsp70 on H1339 and MDA-MB-231 cells, respectively, is associated with a reduced sensitivity to NK cell-mediated lysis. A knockdown of HIF-1α revealed that the decreased surface expression of MICA/B under hypoxia is dependent on HIF-1α in H1339 cells with high basal HIF-1α levels. Hypoxia and HIF-1α did not affect the MICA/B expression in MDA-MB-231 cells but reduced the Hsp70 membrane expression which in turn also impaired NK cell recognition. Furthermore, we could show that the hypoxia-induced decrease in membrane Hsp70 is independent of HIF-1α in MDA-MB-231. Our data indicate that hypoxia-induced downregulation of both NK cell ligands MICA/B and Hsp70 impairs NK cell-mediated cytotoxicity, whereby only MICA/B appears to be regulated by HIF-1α.     

Isoform-specific and Protein Kinase C-mediated Regulation of CTP:Phosphoethanolamine Cytidylyltransferase Phosphorylation

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, -α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50–90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.     

Hsp40 Chaperones Promote Degradation of the hERG Potassium Channel

Loss of function mutations in the hERG (human ether-a-go-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.     

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase β-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, ²⁹⁶EEAMAIAS³⁰⁴, in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to β-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.     

The DNAJA2 Substrate Release Mechanism Is Essential for Chaperone-mediated Folding

DNAJA1 (DJA1/Hdj2) and DNAJA2 (DJA2) are the major J domain partners of human Hsp70/Hsc70 chaperones. Although they have overall similarity with the well characterized type I co-chaperones from yeast and bacteria, they are biologically distinct, and their functional mechanisms are poorly characterized. We identified DJA2-specific activities in luciferase folding and repression of human ether-a-go-go-related gene (HERG) trafficking that depended on its expression levels in cells. Mutations in different internal domains of DJA2 abolished these effects. Using purified proteins, we addressed the mechanistic defects. A mutant lacking the region between the zinc finger motifs (DJA2-Δm2) was able to bind substrate similar to wild type but was incapable of releasing substrate during its transfer to Hsc70. The equivalent mutation in DJA1 also abolished its substrate release. A DJA2 mutant (DJA-221), which had its C-terminal dimerization region replaced by that of DJA1, was inactive but retained its ability to release substrate. The release mechanism required the J domain and ATP hydrolysis by Hsc70, although the nucleotide dependence diverged between DJA2 and DJA1. Limited proteolysis suggested further conformational differences between the two wild-type co-chaperones and the mutants. Our results demonstrate an essential role of specific DJA domains in the folding mechanism of Hsc70.     

Tyrosine Phosphorylation on Spleen Tyrosine Kinase (Syk) Is Differentially Regulated in Human and Murine Platelets by Protein Kinase C Isoforms

Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.     

Co-chaperone CHIP associates with mutant Cu/Zn-superoxide dismutase proteins linked to familial amyotrophic lateral sclerosis and promotes their degradation by proteasomes

Although the ubiquitin-proteasome system and the molecular chaperones are implicated to play an important role in pathogenesis of familial amyotrophic lateral sclerosis (FALS) caused by mutations in Cu/Zn-superoxide dismutase (SOD1), the mechanism underlying the causes of this fatal disease is still poorly understood. Here we found that co-chaperone CHIP (carboxyl terminus of Hsc70-interacting protein), together with molecular chaperones Hsc70/Hsp70 and Hsp90, associates with FALS-linked mutant SOD1 proteins in cultured human cells. S5a subunit of 26S proteasomes, which recognizes polyubiquitylated proteins, also interacts with mutant SOD1 proteins. Over-expression of CHIP leads to the reduction in cellular levels of mutant SOD1 as well as the suppression of cytotoxicity induced by mutant SOD1. Unusually, rather than increasing the level of poly-ubiquitylated SOD1, over-expressed CHIP alters the ubiquitylation pattern of mutant SOD1 proteins. Both down-regulation and ubiquitylation of mutant SOD1 are greatly reduced by a mutant CHIP protein lacking U-box domain. Taken together, these results suggest that co-chaperone CHIP, possibly with another E3 ligase(s), modulates the ubiquitylation of mutant SOD1 and renders them more susceptible for proteasomal degradation.     

Hispolon promotes MDM2 downregulation through chaperone-mediated autophagy

Amplification and overexpression of murine double minute (MDM2) has been observed in several human cancers. Some chemotherapeutic agents cause MDM2 ubiquitination and degradation in a proteasome-dependent system. In addition to the proteasome system, chaperone-mediated autophagy (CMA) is a lysosomal pathway for selective misfolded protein degradation. Molecular chaperone heat shock cognate 70 protein (Hsc70) recognizes the misfolded proteins, which are then delivered to lysosome-associated membrane protein type 2A (LAMP2A) for lysosomal degradation. Our previous study reported that hispolon was able to induce cell apoptosis and downregulate MDM2 expression. In this study, our results showed that the proteasome inhibitor, MG132, could not inhibit hispolon-induced MDM2 downregulation. In contrast, both inhibition of lysosomes with NH₄Cl and inhibition of LAMP2A using siRNA partially attenuated hispolon-induced MDM2 downregulation. To determine whether Hsc70 recognizes MDM2 on amino acids 135-141, SMP14 antibody was used to compete with Hsc70 for interaction with MDM2. After Hsc70 knockdown, SMP14 antibody immunoprecipitated increased MDM2. We also found that hispolon induced increased association of Hsp70, Hsc70, Hsp90 and LAMP2A with MDM2. This association was inhibited in cells pretreated with geldanamycin (GA), an Hsp90 inhibitor. GA also attenuated hispolon-induced MDM2 downregulation. Meanwhile, inhibition of Hsc70 using siRNA attenuated hispolon-induced MDM2 downregulation. Our study provides the first example of the ability of hispolon to mediate MDM2 downregulation in lysosomes through the CMA pathway.     

Down-regulating protein kinase C alpha: Functional cooperation between the proteasome and the endocytic system

Ubiquitination, proteasome, caveolae and endosomes have been implicated in controlling protein kinase Cα (PKCα) down-regulation. However, the molecular mechanism remained obscure. Here we show that endosomes and proteasome cooperate in phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA)-induced down-regulation of PKCα. We show that following TPA treatment and translocation to the plasma membrane, PKCα undergoes multimonoubiquitination prior to its degradation by the proteasome. However, to reach the proteasome, PKCα must travel through the endocytic system from early to late endosomes. This route requires functional endosomes, whereby endosomal alkalinization, or ablation, abrogates completely PKCα degradation maintaining the enzyme at the plasma membrane. This route also depends on synaptotagmin (Syt) II and the Rab7 GTPase, whereby Syt II knock-down or expression of the GDP-locked Rab7 inactive mutant prevents PKCα degradation. We further show that proteasome plays a dual role, where an active proteasome is required for deubiquitination of PKCα, a step crucial to prevent PKCα targeting to the endocytic recycling compartment. Finally, we show that the association with rafts-localized cell surface proteins that internalize in a clathrin-independent fashion is necessary to allow the trafficking of PKCα from the plasma membrane to the proteasome, its ultimate degradation station.     

Disruption of Heat Shock Protein 90 (Hsp90)-Protein Kinase Cδ (PKCδ) Interaction by (?)-Maackiain Suppresses Histamine H1 Receptor Gene Transcription in HeLa Cells

The histamine H₁ receptor (H1R) gene is an allergic disease sensitive gene, and its expression level is strongly correlated with the severity of allergic symptoms. (−)-Maackiain was identified as a Kujin-derived anti-allergic compound that suppresses the up-regulation of the H1R gene. However, the underlying mechanism of H1R gene suppression remains unknown. Here, we sought to identify a target protein of (−)-maackiain and investigate its mechanism of action. A fluorescence quenching assay and immunoblot analysis identified heat shock protein 90 (Hsp90) as a target protein of (−)-maackiain. A pull-down assay revealed that (−)-maackiain disrupted the interaction of Hsp90 with PKCδ, resulting in the suppression of phorbol 12-myristate 13-acetate (PMA)-induced up-regulation of H1R gene expression in HeLa cells. Additional Hsp90 inhibitors, including 17-(allylamino)-17-demethoxygeldanamycin, celastrol, and novobiocin also suppressed PMA-induced H1R gene up-regulation. 17-(Allylamino)-17-demethoxygeldanamycin inhibited PKCδ translocation to the Golgi and phosphorylation of Tyr³¹¹ on PKCδ. These data suggest that (−)-maackiain is a novel Hsp90 pathway inhibitor. The underlying mechanism of the suppression of PMA-induced up-regulation of H1R gene expression by (−)-maackiain and Hsp90 inhibitors is the inhibition of PKCδ activation through the disruption of Hsp90-PKCδ interaction. Involvement of Hsp90 in H1R gene up-regulation suggests that suppression of the Hsp90 pathway could be a novel therapeutic strategy for allergic rhinitis.     

PKCβ Positively Regulates RANKL-Induced Osteoclastogenesis by Inactivating GSK-3β

Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. However, the role of PKC in receptor activator of NF-κB ligand (RANKL) signaling has remained elusive. We now demonstrate that PKCβ acts as a positive regulator which inactivates glycogen synthase kinase-3β (GSK-3β) and promotes NFATc1 induction during RANKL-induced osteoclastogenesis. Among PKCs, PKCβ expression is increased by RANKL. Pharmacological inhibition of PKCβ decreased the formation of osteoclasts which was caused by the inhibition of NFATc1 induction. Importantly, the phosphorylation of GSK-3β was decreased by PKCβ inhibition. Likewise, down-regulation of PKCβ by RNA interference suppressed osteoclast differentiation, NFATc1 induction, and GSK-3β phosphorylation. The administration of PKC inhibitor to the RANKL-injected mouse calvaria efficiently protected RANKL-induced bone destruction. Thus, the PKCβ pathway, leading to GSK-3β inactivation and NFATc1 induction, has a key role in the differentiation of osteoclasts. Our results also provide a further rationale for PKCβ’s therapeutic targeting to treat inflammation-related bone diseases.     

Human heat shock protein 105/110 kDa (Hsp105/110) regulates biogenesis and quality control of misfolded cystic fibrosis transmembrane conductance regulator at multiple levels

Heat shock protein 105/110-kDa (Hsp105/110), a member of the Hsp70 super family of molecular chaperones, serves as a nucleotide exchange factor for Hsc70, independently prevents the aggregation of misfolded proteins, and functionally relates to Hsp90. We investigated the roles of human Hsp105α, the constitutively expressed isoform, in the biogenesis and quality control of the cystic fibrosis transmembrane conductance regulator (CFTR). In the endoplasmic reticulum (ER), Hsp105 facilitates CFTR quality control at an early stage in its biosynthesis but promotes CFTR post-translational folding. Deletion of Phe-508 (ΔF508), the most prevalent mutation causing cystic fibrosis, interferes with de novo folding of CFTR, impairing its export from the ER and accelerating its clearance in the ER and post-Golgi compartments. We show that Hsp105 preferentially associates with and stabilizes ΔF508 CFTR at both levels. Introduction of the Hsp105 substrate binding domain potently increases the steady state level of ΔF508 CFTR by reducing its early-stage degradation. This in turn dramatically enhances ΔF508 CFTR cell surface functional expression in cystic fibrosis airway epithelial cells. Although other Hsc70 nucleotide exchange factors such as HspBP1 and BAG-2 inhibit CFTR post-translational degradation in the ER through cochaperone CHIP, Hsp105 has a primary role promoting CFTR quality control at an earlier stage. The Hsp105-mediated multilevel regulation of ΔF508 CFTR folding and quality control provides new opportunities to understand how chaperone machinery regulates the homeostasis and functional expression of misfolded proteins in the cell. Future studies in this direction will inform therapeutics development for cystic fibrosis and other protein misfolding diseases.     

Functional Role for Protein Kinase Cβ as a Regulator of Stress-Activated Protein Kinase Activation and Monocytic Differentiation of Myeloid Leukemia Cells

Human myeloid leukemia cells respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and other activators of protein kinase C (PKC) with induction of monocytic differentiation. The present studies demonstrated that treatment of U-937 and HL-60 myeloid leukemia cells with TPA, phorbol-12,13-dibutyrate, or bryostatin 1 was associated with the induction of stress-activated protein kinase (SAPK). In contrast, TPA-resistant TUR and HL-525 cell variants deficient in PKCβ failed to respond to activators of PKC with the induction of SAPK. A direct role for PKCβ in TPA-induced SAPK activity in TUR and HL-525 cells that stably express PKCβ was confirmed. We showed that TPA induced the association of PKCβ with MEK kinase 1 (MEKK-1), an upstream effector of the SAPK/ERK kinase 1 (SEK1)→SAPK cascade. The results also demonstrated that PKCβ phosphorylated and activated MEKK-1 in vitro. The functional role of MEKK-1 in TPA-induced SAPK activity was further supported by the demonstration that the expression of a dominant negative MEKK-1 mutant abrogated this response. These findings indicate that PKCβ activation is necessary for activation of the MEKK-1→SEK1→SAPK cascade in the TPA response of myeloid leukemia cells.     

Expression of Protein Kinase C Isoforms in Pancreatic Islets and Liver of Male Goto-Kakizaki Rats,a Model of Type 2 Diabetes

Protein kinase C (PKC) is a family of protein kinases controlling protein phosphorylation and playing important roles in the regulation of metabolism. We have investigated expression levels of PKC isoforms in pancreatic islets and liver of diabetic Goto-Kakizaki (GK) rats with and without insulin treatment to evaluate their association with glucose homeostasis. mRNA and protein expression levels of PKC isoforms were assessed in pancreatic islets and liver of Wistar rats and GK rats with or without insulin treatment. PKCα and PKCζ mRNA expressions were down-regulated in islets of GK compared with Wistar rats. PKCα and phosphorylated PKCα (p-PKCα) protein expressions were decreased in islets of GK compared with insulin-treated GK and Wistar rats. PKCζ protein expression in islets was reduced in GK and insulin-treated GK compared with Wistar rats, but p-PKCζ was decreased only in GK rats. Islet PKCε mRNA and protein expressions were lower in GK compared with insulin-treated GK and Wistar rats. In liver, PKCδ and PKCζ mRNA expressions were decreased in both GK and insulin-treated GK compared with Wistar rats. Hepatic PKCζ protein expression was diminished in both GK rats with and without insulin treatment compared with Wistar rats. Hepatic PKCε mRNA expression was down-regulated in insulin-treated GK compared with GK and Wistar rats. PKCα, PKCε, and p-PKCζ expressions were secondary to hyperglycaemia in GK rat islets. Hepatic PKCδ and PKCζ mRNA expressions were primarily linked to hyperglycaemia. Additionally, hepatic PKCε mRNA expression could be under control of insulin.