Beta Isoform Of Protein Kinase Research Articles

Protein Kinase A isoform dynamics and disease.

A number of disease mutations in the alpha and beta isoforms of Protein Kinase A have recently been identified. We model these mutations and investigate changes in conformational and protein/ligand interaction dynamics. Changes in partner protein and substrate interactions due to mutation is confirmed by fluorescence polarization binding and competition assays. Computational and biochemical approaches reveal a potential common mechanism among the mutations leading to kinase dysfunction.

A randomized, double-blind, placebo-controlled, Phase II study with and without enzastaurin in combination with docetaxel-based chemotherapy in patients with castration-resistant metastatic prostate cancer

Enzastaurin is an oral serine/threonine kinase inhibitor that inhibits the beta isoform of protein kinase C and which may have therapeutic activity in prostate cancer. We explored the efficacy of docetaxel/prednisone with or without enzastaurin in patients with castration-resistant metastatic prostate cancer. A nonrandomized safety cohort consisting of 14 patients was followed by a double-blind randomized Phase II trial. Patients received standard doses of docetaxel (75 mg/m(2)) with prednisone 10 mg daily with or without 500 mg/day of enzastaurin. There was no difference in the objective response rate between the enzastaurin and placebo arms (placebo: 7 [15.2 %]; enzastaurin: 6 [15.0 %]; P = 1.00). The median PFS was 229 days for patients in the enzastaurin arm versus 213 days for the placebo arm (P = 0.524). The 1-year overall survival rates were almost identical, with 76.7 % and 75.1 % in the enzastaurin and placebo arms, respectively. Therapy was well tolerated although the combination of enzastaurin and docetaxel was more myelosuppressive than with docetaxel alone. The clinical activity of docetaxel/prednisone plus enzastaurin cannot be distinguished from docetaxel/prednisone alone, given the limitations of a randomized Phase II design. Although the toxicity profile was favorable for the enzastaurin-containing regimen, there is no compelling rationale to move this combination forward for the treatment of castration-resistant metastatic prostate cancer.

Oral enzastaurin in prostate cancer: A two-cohort phase II trial in patients with PSA progression in the non-metastatic castrate state and following docetaxel-based chemotherapy for castrate metastatic disease

Enzastaurin is an oral serine/threonine kinase inhibitor of the beta isoform of protein kinase C that may have therapeutic activity in prostate cancer. We explored the efficacy of enzastaurin on two cohorts of patients with prostate cancer progression in the castrate state. A two-cohort phase II trial was conducted, with both groups participating simultaneously. Cohort 1 consisted of patients with non-metastatic castrate prostate-specific antigen progressive disease. Cohort 2 consisted of patients with castrate metastatic disease with progression following docetaxel-based chemotherapy. Patients in both cohorts received 500 mg/day enzastaurin. Therapy was well tolerated in both cohorts. One complete response was observed in Cohort 1, with limited activity in the majority of patients. In Cohort 2, no objective responses were seen and the median progression-free survival (11 weeks [90% confidence interval: 7.6, 11.7]) did not differ from the historical control. Enzastaurin as a single agent has limited activity in castrate progressive prostate cancer. Evaluation in combination with docetaxel is ongoing.

Ruboxistaurin attenuates hypertriglyceridemia in diabetic rats: Comparison with the antioxidant N‐acetylcysteine

Hyperglycemia‐induced oxidative stress plays a critical role in the development of diabetic metabolic complications including hypertriglyceridemia. The beta isoform of Protein kinase C (PKCβ) is over‐expressed in various organs of diabetic rodents and can cause or exacerbate oxidative stress by activating NADPH oxidase, a major source of superoxide production in the cardiovascular system. We postulated that selective PKCβ inhibition with ruboxistaurin (RXT) may attenuate hypertriglyceridemia in diabetes by reducing oxidative stress. Control or streptozotozin‐induced diabetic rats were either untreated (C, D) or treated with RXT (1 mg/kg/day, D+RXT) or with the antioxidant N‐acetylcycsteine (NAC) (1.5g/kg/day, D+NAC) delivered by oral gavage for four weeks. Levels of 15‐F2t‐isoprostane (IsoP), a specific marker of oxidative stress, were significantly increased in D group in both the myocardium and plasma. This was accompanied by a marked increase in plasma triglycerides (P<0.05 vs. C). RXT and NAC, respectively, prevented the increase of IsoP and significantly attenuated the increases of triglycerides. However, triglycerides in the D+NAC group was lowered further than that in the D+RXT group (P<0.05). Despite similar antioxidant properties, ruboxistaurin seems to be inferior to NAC in reducing hypertriglyceridemia in diabetes.Supported by RGC/GRF grants (781109 M to ZX, 766709 M to MGI).

PKC β inhibitor ruboxistaurin prevents the increase of 15‐F2t‐isoprostane in the myocardium and plasma in Type 1 diabetic rats

Levels of 15‐F2t‐isoprostane (IsoP), a specific marker of oxidative stress and a recently identified independent predictor of myocardial morbidity in patients with ischemic heart disease, are increased in the myocardium and plasma in Type 1 diabetes. The beta isoform of Protein kinase C (PKCβ) is over‐expressed in the myocardium of diabetic rodents and contributes to the development of diabetic cardiomyopathy and associated complications. We postulated that selective PKCâ inhibition with ruboxistaurin would attenuate hyperglycemia‐induced oxidative stress and normalize IsoP production. Control or streptozotozin‐induced diabetic rats were treated (DT) or untreated (C, D) with ruboxistaurin (1 mg/kg/day) delivered by oral gavage for four weeks. Myocardial IsoP content, cardiac mass and plasma IsoP were significantly increased while there was a significant decrease in superoxide dismutase activity in D compared to C rats (all P<0.05). Ruboxistaurin treatment normalized all these changes. It is concluded that ruboxistaurin may have prevented hyperglycemia‐induced oxidative stress by restoring endogenous antioxidant enzyme superoxide dismutase activity in diabetes.Supported by RGC/GRF grants (HKU781109M to ZX, HKU766709M to MGI) and University of Hong Kong seeding grant for basic research (ZX).

Ruboxistaurin mesilate hydrate for diabetic retinopathy

Diabetic retinopathy remains a major worldwide cause of preventable blindness. The beta isoform of protein kinase C (PKC) may play an important role in the pathogenesis of this disorder. Ruboxistaurin mesylate hydrate is an orally bioavailable, highly-specific inhibitor of PKC beta, which has shown some efficacy in several large, multicenter, randomized clinical trials. The U.S. Food and Drug Administration issued an approvable letter for ruboxistaurin in 2006, but at this time the medication is not available for routine clinical use.

Disposition of [14C]Ruboxistaurin in Humans

Ruboxistaurin is a potent and specific inhibitor of the beta isoforms of protein kinase C (PKC) that is being developed for the treatment of diabetic microvascular complications. The disposition of [(14)C]ruboxistaurin was determined in six healthy male subjects who received a single oral dose of 64 mg of [(14)C]ruboxistaurin in solution. There were no clinically significant adverse events during the study. Whole blood, urine, and feces were collected at frequent intervals after dosing. Metabolites were profiled by high performance liquid chromatography with radiometric detection. The total mean recovery of the radioactive dose was approximately 87%, with the majority of the radioactivity (82.6 +/- 1.1%) recovered in the feces. Urine was a minor pathway of elimination (4.1 +/- 0.3%). The major route of ruboxistaurin metabolism was to the N-desmethyl ruboxistaurin metabolite (LY338522), which has been shown to be active and equipotent to ruboxistaurin in the inhibition of PKC(beta). In addition, multiple hydroxylated metabolites were identified by liquid chromatography-mass spectrometry in all matrices. Pharmacokinetics were conducted for both ruboxistaurin and LY338522 (N-desmethyl ruboxistaurin, 1). These moieties together accounted for approximately 52% of the radiocarbon measured in the plasma. The excreted radioactivity was profiled using radiochromatography, and approximately 31% was structurally characterized as ruboxistaurin or N-desmethyl ruboxistaurin. These data demonstrate that ruboxistaurin is metabolized primarily to N-desmethyl ruboxistaurin (1) and multiple other oxidation products, and is excreted primarily in the feces.

Ruboxistaurin

Ruboxistaurin is a potent and specific inhibitor of the beta isoform of protein kinase C. Overactivation of protein kinase C has been demonstrated in patients with type 2 diabetes, and is postulated to play a major role in the pathogenesis of diabetic microvascular complications, which include diabetic retinopathy, neuropathy and nephropathy. The role of protein kinase C in promoting tissue injury in patients with diabetes, and the pharmacologic and clinical studies illustrating the potential of ruboxistaurin to reduce the burden of diabetic microvascular complications will be discussed in this article.

Identification of protein kinase C (PKC) isoforms in teleostean, amphibian and avian pigment cells.

The beta isoform of protein kinase C (PKC) has been described as the main isoform involved in the stimulation of melanogenesis in mammalian skin melanocytes. Little is known about PKC isoforms in non-mammalian pigment cells. In neopterigian fish (holostei and teleostei), PKC is associated with pigment granule aggregation within the pigment cells (skin lightening), whereas in elasmobranchs and tetrapods, the activation of PKC leads to pigment granule dispersion (skin darkening). In an attempt to a better understanding of this distinct functional behavior upon PKC activation, we decided to investigate the PKC isoforms expressed in pigment cell lines of teleost fish, amphibians and birds, using RT-PCR followed by cloning and sequencing. Our results demonstrate the presence of messenger RNA (mRNA) for the following PKC isoforms: beta 1, lambda and iota in GEM-81 cells (Carassius auratus erythrophoroma), beta 1, beta 2 and zeta in Xenopus laevis (amphibian) melanophores; beta 1 and lambda in Gallus gallus (chicken) primary melanocytes. Beta 1 PKC seems to be conserved throughout phylogeny, but the diversity of the other isoforms in the different groups may account for the functional differences after PKC activation, which are observed between teleost and tetrapod pigment cells.

Is there a link between impaired glucose metabolism and protein kinase C activity in the diabetic heart?

The activity of the beta isoform of protein kinase C (PKC beta) is reduced in the diabetic heart. Since this isozyme has been implicated in insulin action, we tested the hypothesis that PKC beta contributes to the development of impaired glucose metabolism by the noninsulin-dependent diabetic heart. Exposure of the diabetic heart to buffer containing the protein kinase C activator, phorbol myristate acetate, increased PKC beta activity in the membrane. Associated with the improvement in PKC beta activity was a biphasic change in glucose metabolism. The initial phase was characterized by a breakdown in glycogen stores, a stimulation in glucose oxidation and a decrease in endogenous fatty acid oxidation. This was followed by a second phase in which the uptake of glucose was modestly stimulated. Nonetheless, since the phorbol ester did not overcome the diabetes-linked defect in pyruvate dehydrogenase, the increase in glycolytic flux was not associated with a rise in glucose oxidation. Consequently, nearly 50% of the triose units were diverted into lactate and pyruvate production and the generation of ATP from glucose was restricted. Since insulin promotes not only glucose uptake, but also glycogen synthesis and glucose oxidation, the phorbol ester and insulin effects are very different. Thus, the data do not support a role for PKC beta in the development of glucose metabolic defects in the hearts of noninsulin-dependent diabetic rats.

The beta isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells

We have investigated the role of protein kinase C (PKC) in human melanogenesis. The level of PKC activity paralleled the total melanin content in cultured newborn melanocytes. Activation of PKC by treatment with 5 x 10(-7) M phorbol dibutyrate acutely caused a doubling in the activity of tyrosinase, the rate-limiting enzyme in melanogenesis, known to correlate directly with melanin synthesis in these cells. When PKC was depleted to 5-10% of initial levels, there was a 40-50% parallel reduction in tyrosinase activity; and regeneration of PKC activity was associated with the recovery of tyrosinase activity. By Northern blot analysis, the alpha and beta but not the gamma isoforms were detectable in melanocytes. By Western blot analysis, the racially determined pigment level in cultured melanocytes correlated with PKC-beta protein expression. In a pigmented human melanoma line (P-MM4, 20-30 ng melanin/micrograms protein)and its nonpigmented subclone (NP-MM4, undetectable melanin), PKC-alpha mRNA was expressed in both, whereas PKC-beta mRNA was detectable only in P-MM4 cells. Tyrosinase protein level was comparable in both cell lines. When NP-MM4 cell lysate was incubated with melanocyte lysate known to contain PKC-beta, tyrosinase activity per microgram of melanocyte protein in the combined lysate increased, consistent with activation of the previously inactive tyrosinase of NP-MM4 origin. Moreover, NP-MM4 cells transiently transfected with PKC-beta cDNA increased tyrosinase activity from undetectable to detectable levels. These combined data show that PKC-beta regulates human melanogenesis by activating tyrosinase.

Interferon-alpha selectively activates the beta isoform of protein kinase C through phosphatidylcholine hydrolysis.

The early events that occur after interferon binds to discrete cell surface receptors remain largely unknown. Human leukocyte interferon (interferon-alpha) rapidly increases the binding of [3H]phorbol dibutyrate to intact HeLa cells (ED50 = 100 units/ml), a measure of protein kinase C activation, and induces the selective translocation of the beta isoform of protein kinase C from the cytosol to the particulate fraction of HeLa cells. The subcellular distribution of the alpha and epsilon isoforms is unaffected by interferon-alpha treatment. Activation of protein kinase C by phorbol esters mimics the inhibitory action of interferon-alpha on HeLa cell proliferation and down-regulation of protein kinase C blocks the induction of antiviral activity by interferon-alpha in HeLa cells. Increased phosphatidylcholine hydrolysis and phosphorylcholine production is accompanied by diacylglycerol production in response to interferon. However, inositol phospholipid turnover and free intracellular calcium concentration are unaffected. These results suggest that the transient increase in diacylglycerol, resulting from phosphatidylcholine hydrolysis, may selectively activate the beta isoform of protein kinase C. Moreover, the activation of protein kinase C is a necessary element in interferon action on cells.

Differential effects of phorbol ester on the in vitro invasiveness of malignant and non‐malignant human fibroblast cells

The effect of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cell invasion was studied using an in vitro assay for cell invasion through a reconstituted basement membrane matrix (Matrigel). TPA inhibited the invasiveness of malignant human fibrosarcoma HT1080 cells. In contrast, WI-38 lung fibroblasts, which show a very low invasive capacity, were stimulated (3-fold) to invade Matrigel after exposure to TPA for 48 hours. The inhibitory or stimulatory effects of TPA on cell invasion were correlated with a decrease or an increase in cell motility and collagenase IV activity, respectively. Synthetic diacylglycerols partially mimicked the inhibitory action of TPA on HT1080 cells but failed to stimulate WI-38 cell invasion. Immunoblots demonstrated that in both cell lines the alpha and beta isoforms of protein kinase C were equally down-regulated after a 5 hour exposure to TPA despite the basal low level of protein kinase C polypeptide in the malignant cells. Thus, whereas in WI-38 cells induction of an invasive behavior could be observed in the absence of protein kinase C, in the malignant cells disappearance of the kinase was associated with a non-invasive phenotype.

Activation of Protein Kinase C by Naturally Occurring Ether-linked Diglycerides

Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.