Increased expression of a high molecular weight (130 KD) protein kinase C isoform in a differentiation-defective ras-transfected keratinocyte line

Among its many biological roles, fibroblast growth factor-2 (FGF2) acutely protects the heart from dysfunction associated with ischemia/reperfusion (I/R) injury. Our laboratory has demonstrated that this is due to the activity of the low molecular weight (LMW) isoform of FGF2 and that FGF2-mediated cardioprotection relies on the activity of protein kinase C (PKC); however, which PKC isoforms are responsible for LMW FGF2-mediated cardioprotection, and their downstream targets, remain to be elucidated. To identify the PKC pathway(s) that contributes to postischemic cardiac recovery by LMW FGF2, mouse hearts expressing only LMW FGF2 (HMWKO) were bred to mouse hearts not expressing PKCα (PKCαKO) or subjected to a selective PKCε inhibitor (εV1–2) before and during I/R. Hearts only expressing LMW FGF2 showed significantly improved postischemic recovery of cardiac function following I/R ( P < 0.05), which was significantly abrogated in the absence of PKCα ( P < 0.05) or presence of PKCε inhibition ( P < 0.05). Hearts only expressing LMW FGF2 demonstrated differences in actomyosin ATPase activity as well as increases in the phosphorylation of troponin I and T during I/R compared with wild-type hearts; several of these effects were dependent on PKCα activity. This evidence indicates that both PKCα and PKCε play a role in LMW FGF2-mediated protection from cardiac dysfunction and that PKCα signaling to the contractile apparatus is a key step in the mechanism of LMW FGF2-mediated protection against myocardial dysfunction.

Download Full-text

High molecular weight FGF-2 promotes postconditioning-like cardioprotection linked to activation of the protein kinase C isoforms Akt and p70 S6 kinaseThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y09-049 ◽

2009 ◽

Vol 87 (10) ◽

pp. 798-804 ◽

Cited By ~ 18

Author(s):

Zhi-Sheng Jiang ◽

Ge-Bo Wen ◽

Zhi-Han Tang ◽

Wattamon Srisakuldee ◽

Robert R. Fandrich ◽

...

Keyword(s):

Molecular Weight ◽

Protein Kinase C ◽

Protein Kinase ◽

Cytochrome C ◽

Contractile Function ◽

Ischemia Reperfusion ◽

Systolic Pressure ◽

Treated Group ◽

Kinase C ◽

Cytosolic Cytochrome

Fibroblast growth factor 2 (FGF-2) is a multifunctional protein translated as high and low molecular weight isoforms (hi- and lo-FGF-2, respectively). Although the postconditioning cardioprotective effect of lo-FGF-2 (18 kDa) has been documented, hi-FGF-2 is less well studied. We used an isolated perfused rat heart model of ischemia–reperfusion to study the effects of postischemic (during reperfusion) administration of hi-FGF-2 on recovery of contractile function and tissue salvage, as indicated by decreased cytosolic cytochrome c levels. Compared with the vehicle-treated group, hi-FGF-2-treated hearts had significantly improved recovery of systolic pressure, developed pressure, rates of contraction and relaxation, and coronary flow, as well as decreased relative levels of cytosolic cytochrome c. The effects of hi-FGF-2 on functional recovery and cytosolic cytochrome c were indistinguishable from those induced by lo-FGF-2. Both hi- and lo-FGF-2 upregulated relative levels of phosphorylated (activated) Akt and p70 S6 kinase, and they both promoted translocation of α, ε, and ζ isoforms of protein kinase C (PKC) to the particulate fraction of reperfused hearts. The magnitude of the effect on PKCζ and p70 S6 kinases, however, was significantly more potent in the hi-FGF-2 than in the lo-FGF-2 group. We conclude that acute postischemic cardioprotection by hi- or lo-FGF-2 is isoform nonspecific and likely to be mediated by PKC and Akt. Nevertheless, isoform-specific functions are suggested by the augmented sensitivity of p70 S6 and PKCζ to hi-FGF-2.

Download Full-text

Reduced protein kinase C delta association with a higher molecular weight complex in mitochondria of Barth Syndrome lymphoblasts

10.1101/2021.07.21.453087 ◽

2021 ◽

Author(s):

Edgard M Mejia ◽

Hana M Zegallai ◽

Genevieve C Sparagna ◽

Grant M Hatch

Keyword(s):

Molecular Weight ◽

Protein Kinase C ◽

Protein Kinase ◽

Oxidative Phosphorylation ◽

Barth Syndrome ◽

Mitochondrial Oxidative Phosphorylation ◽

High Molecular Weight Complex ◽

Protein Kinase C Delta ◽

Kinase C ◽

Mitochondrial Fractions

Protein kinase C delta (PKCδ) is a signaling kinase that regulates several cellular responses and is controlled via multi-site phosphorylation. The PKCδ signalosome exists as a high molecular weight complex in mitochondria and adjusts the fuel flux from glycolytic sources to the intensity of mitochondrial respiration, thus controlling mitochondrial oxidative phosphorylation. In the X-linked genetic disease Barth Syndrome (BTHS) mitochondrial oxidative phosphorylation is impaired due to a mutation in the TAFAZZIN gene which results in reduction in the phospholipid cardiolipin (CL) and an accumulation of monolysocardiolipin (MLCL). We previously demonstrated, through phosphokinome analysis, that phosphorylation of PKCδ is altered on multiple sites in BTHS patient lymphoblasts. In this study, we examined if PKCδ association with a higher molecular weight complex was altered in mitochondria of BTHS lymphoblasts. BTHS lymphoblasts exhibited reduction in all molecular species of CL examined and accumulation of trioleoyl-MLCL. Immunoblot analysis of blue native-polyacrylamide gel electrophoresis mitochondrial fractions revealed that PKCδ was associated with a higher molecular weight complex and that this was markedly reduced in BTHS patient B lymphoblasts compared to controls in spite of an increase in PKCδ protein expression. The elevated expression level of PKCδ in BTHS lymphoblasts was associated with increased citrate synthase activity indicative of abnormal mitochondrial proliferation. We hypothesize that the lack of PKCδ within this higher molecular weight complex may contribute to defective mitochondrial PKCδ signaling and thus to the bioenergetic defects observed in BTHS.

Download Full-text

ENZYMES THAT DEPHOSPHORYLATE INOSITOL PHOSPHATES IN HUMAN PLATELETS

10.1055/s-0038-1644522 ◽

1987 ◽

Author(s):

Luis Molina ◽

y Vedia ◽

Eduardo G Lapetina

Keyword(s):

Molecular Weight ◽

Protein Kinase C ◽

Protein Kinase ◽

Membrane Fraction ◽

Inositol Phosphates ◽

Human Platelets ◽

Particulate Fraction ◽

Cytosolic Fraction ◽

Specific Marker ◽

Kinase C

Inositol trisphosphate (IP3) is now recognized as a second messenger molecule that mobilizes Ca2+ from intracellular stores to the cytosol. The persistence of the action of IP3 depends on the specific phosphatase that converts IP3 to inositol bisphosphate (IP2). The activation of IP3 phosphatase is important in terminating the Ca2+ signal in stimulated cells. In platelets it has previously been shown that this enzyme is regulated by protein kinase C since it is stimulated by phorbol esters and 1,2-diacylglycerol (Molina y Vedia, L., and Lapetina, E.G. J. Biol Chem. 261, 10493-10495, 1986) and the cytosolic platelet enzyme is phosphorylated by brain protein kinase C, resulting in a 4-fold increase in IP3 phosphatase activity (Connolly, T. M., Lawing, W.J., Jr., and Majerus, P.W., Cell, 46, 951-958, 1986). We have studied the subcellular distribution of the phosphatases that hydrolyze IP3, IP2 and inositol monophosphate (IP) in human platelets. Three subcellular fractions were obtained from human platelets lysed by freezing and thawing: a cytosolic fraction, a membrane fraction and a mixed particulate fraction containing granules, mitochondria and membranes. These fractions have been characterized by specific marker enzymes. The highest specific activity of IP3 -phosphatase is associated with the membrane fraction and accounts for about 10-15% of the total activity. The mixed particulate fraction has 35-40% of the activity while about 50% is cytosolic. The Km of the membrane fraction enzyme is 100 μM. This enzyme is extracted by 1M NaCl and hydrodynamic studies revealed a molecular weight of 50 kDa. The NaCl extracted-enzyme has been further purified by hydrophobic and gel filtration chromatographies. This activity does not hydrolyses IP but hydrolyse IP2 at a lower rate. The enzyme that hydrolyses IP to inositol is confined to the cytosolic fraction, has a Km of 130 μM, is inhibited by Li+, and hydrodynamic studies show an apparent molecular weight of 91 kDa.

Download Full-text

Pentobarbital, but not Propofol, Suppresses Vasopressin-stimulated Heat Shock Protein 27 Induction in Aortic Smooth Muscle Cells

Anesthesiology ◽

10.1097/00000542-200006000-00041 ◽

2000 ◽

Vol 92 (6) ◽

pp. 1807-1813 ◽

Cited By ~ 5

Author(s):

Osamu Kozawa ◽

Kumiko Tanabe ◽

Hiroyuki Matsuno ◽

Masayuki Niwa ◽

Takuji Yamamoto ◽

...

Keyword(s):

Molecular Weight ◽

Smooth Muscle ◽

Protein Kinase C ◽

Heat Shock ◽

Protein Kinase ◽

Heat Shock Protein ◽

Smooth Muscle Cells ◽

Muscle Cells ◽

Aortic Smooth Muscle ◽

Kinase C

Background Although it is known that systemic blood pressure decreases after the administration of pentobarbital or propofol, the mechanisms underlying the cardiovascular effects of these anesthetics are still poorly understood. The authors previously showed that vasopressin stimulates the induction of heat shock protein (HSP) 27, a low-molecular-weight HSP, by a protein kinase C-dependent manner in aortic smooth muscle A10 cells. It is recognized that HSP27 may act as a chaperone like high-molecular-weight HSPs such as HSP70. HSP27 is reportedly associated with agonist-induced contraction of vascular smooth muscle cells. The authors examined the effects of pentobarbital and propofol on the vasopressin-stimulated HSP27 induction in A10 cells. Methods Cultured A10 cells were pretreated with pentobarbital or propofol and then stimulated by vasopressin or 12-o-tetradecanoylphorbol 13-acetate (TPA). The effect of vasopressin on HSP70 was evaluated by Western blot analysis and compared with its effect on HSP27. The concentrations of HSP27 were determined by a specific immunoassay. The effect of pentobarbital on the expression levels of mRNA for HSP27 by vasopressin was evaluated by Northern blot analysis. Results Vasopressin induced HSP27 but had little effect on HSP70. At concentrations used clinically, pentobarbital inhibited the accumulation of HSP27 by vasopressin or TPA. Pentobarbital reduced the levels of mRNA for HSP27 induced by vasopressin. In contrast, propofol affected neither the vasopressin- nor TPA-induced HSP27 accumulation. Conclusions These results suggest that pentobarbital suppresses the vasopressin-stimulated HSP27 induction in vascular smooth muscle cells. This inhibitory effect is probably exerted at a point downstream from protein kinase C.

Download Full-text