Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites

To determine the in vivo functional significance of troponin I (TnI) protein kinase C (PKC) phosphorylation sites, we created a transgenic mouse expressing mutant TnI, in which PKC phosphorylation sites at serines-43 and -45 were replaced by alanine. When we used high-perfusate calcium as a PKC activator, developed pressures in transgenic (TG) perfused hearts were similar to wild-type (WT) hearts ( P = not significant, NS), though there was a 35% and 32% decrease in peak-systolic intracellular calcium ( P < 0.01) and diastolic calcium ( P < 0.005), respectively. The calcium transient duration was prolonged in the TG mice also (12–27%, ANOVA, P < 0.01). During global ischemia, TG hearts developed ischemic contracture to a greater extent than WT hearts (41 ± 18 vs. 69 ± 10 mmHg, perfusate calcium 3.5 mM, P < 0.01). In conclusion, expression of mutant TnI lacking PKC phosphorylation sites results in a marked alteration in the calcium-pressure relationship, and thus susceptibility to ischemic contracture. The reduced intracellular calcium and prolonged calcium transients suggests that a potent feedback mechanism exists between the myofilament and the processes controlling calcium homeostasis.

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Abstract 5403: Protein Kinase C-Induced Troponin I Phosphorylation Causes Changes in Cardiac Contractile Function but not the Intracellular Calcium Transient

Circulation ◽

10.1161/circ.118.suppl_18.s_544 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Jonathan A Kirk ◽

Stephen H Smith ◽

Guy A MacGowan ◽

Sanjeev G Shroff

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Intracellular Calcium ◽

Cardiac Muscle ◽

Troponin I ◽

Contractile Function ◽

Calcium Transient ◽

Kinase C ◽

Intracellular Calcium Transient ◽

Muscle Contractile

Both intracellular calcium transients ([Ca] i ) and myofilament properties determine cardiac muscle contractile force. Transgenic mouse models created to perturb specific myofilament proteins often cause a compensatory change in [Ca] i , which confounds the assessment of myofilament structure-function relationships. We have created a new transgenic mouse that has all three protein kinase C (PKC) phosphorylation sites on cardiac troponin I (cTnI) mutated to glutamic acid, rendering these sites constitutively pseudo-phosphorylated. Our goal was to determine the effects of this mutation on cardiac muscle contractile function and whether these effects would be concurrent with changes in the [Ca] i . Two sets of studies were conducted: skinned muscle fiber experiments to characterize the steady-state force-pCa relationships at sarcomere lengths of 1.9 and 2.3 μm and right ventricular papillary muscle experiments to characterize the peak developed force (F dev )-muscle length (L) relationships and [Ca] i (fura-5F calcium dye, emission: 510 nm, excitation: 340 and 380 nm, R = [emission fluorescence 340 ]/[emission fluorescence 380 ]). In skinned fibers, there was a significant decrease in maximally activated force (i.e., force at pCa 4.33) in transgenic mice (Wild-Type, WT (n = 7): 64.4± 8.0, Transgenic, TG (n = 6): 42.6±6.8 mN•mm −2 , P = 0.004), without any changes in calcium sensitivity or cooperativity (Hill coefficient). In intact papillary muscles, TG mice showed a decrease in F dev and slowed relaxation for all muscle lengths examined (F dev @ 100% L max , WT (n = 5): 9.3±3.5, TG (n = 6): 4.2±1.6 mN•mm −2 , P = 0.005; dF/dt min @ 100% L max , WT: −136±32, TG: −74±38 mN•mm −2 •s −1 , P = 0.002). In contrast, [Ca] i was unaltered in TG mice at all muscle lengths examined ([Ca] i amplitude as quantified by R systole / R diaastole , WT: 1.62±0.07, TG: 1.48±0.22; [Ca] i relaxation rate d R /dt min , WT: −96±37, TG: −64±30 s −1 ). Thus, PKC-induced TnI phosphorylation affects cardiac muscle contraction (reduced force magnitude and slowed relaxation) via changes in the myofilament properties (activation and/or crossbridge dynamics), and these contractile effects are not related to any changes in the intracellular calcium transient.

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Identification of sites phosphorylated in bovine cardiac troponin I and troponin T by protein kinase C and comparative substrate activity of synthetic peptides containing the phosphorylation sites

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)47130-5 ◽

1989 ◽

Vol 264 (34) ◽

pp. 20778-20785

Author(s):

T.A. Noland ◽

R.L. Raynor ◽

J.F. Kuo

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cardiac Troponin ◽

Synthetic Peptides ◽

Troponin I ◽

Troponin T ◽

Cardiac Troponin I ◽

Phosphorylation Sites ◽

Kinase C

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Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C

Biochemical Journal ◽

10.1042/bj3140937 ◽

1996 ◽

Vol 314 (3) ◽

pp. 937-942 ◽

Cited By ~ 16

Author(s):

Karen L. CRAIG ◽

Calvin B. HARLEY

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phosphorylation Sites ◽

Wild Type ◽

Cos Cells ◽

Phospholipid Hydrolysis ◽

Kinase C

During platelet activation, receptor-coupled phospholipid hydrolysis stimulates protein kinase C (PKC) and results in the phosphorylation of several proteins, the most prominent being pleckstrin. Pleckstrin is composed of two repeated domains, now called pleckstrin homology (PH) domains, separated by a spacer region that contains several consensus PKC phosphorylation sites. To determine the role of PKC-dependent phosphorylation in pleckstrin function, we mapped the phosphorylation sites in vivo of wild-type and site-directed mutants of pleckstrin expressed in COS cells. Phosphorylation was found to occur almost exclusively on Ser-113 and Ser-117 within the sequence 108-KFARKS*TRRS*IRL-120. Phosphorylation of these sites was confirmed by phosphorylation of the corresponding wild-type and mutant synthetic peptides in vitro.

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Troponin I protein kinase C phosphorylation sites and ventricular function

Cardiovascular Research ◽

10.1016/j.cardiores.2004.04.010 ◽

2004 ◽

Vol 63 (2) ◽

pp. 245-255 ◽

Cited By ~ 20

Author(s):

G MACGOWAN

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Ventricular Function ◽

Troponin I ◽

Phosphorylation Sites ◽

Kinase C

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Apoptin Induced Tumour Specific Killing Via Protein Kinase C Isoforms: A Potential Therapeutic Strategy In Plasma Cell Dyscrasias

Blood ◽

10.1182/blood.v116.21.5039.5039 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5039-5039

Author(s):

Jie Jiang ◽

Daryl Cole ◽

Nigel Westwood ◽

Lee Macpherson ◽

Farzin Farzaneh ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Protein Kinase C ◽

Protein Kinase ◽

Cell Line ◽

Phosphorylation Sites ◽

Normal Cells ◽

Myeloma Cells ◽

Kinase C

Abstract Abstract 5039 There is mounting evidence that malignant cells have an intrinsic ability to prevent apoptosis. In the present study we provide evidence that the ectopic expression of Apoptin can restore the failing apoptosis program in myeloma cells via protein kinase C b (PKCb) and overcome intrinsic or acquired resistance to cell death. Apoptin (VP3), a chicken anemia virus (CAV)-derived protein has been shown to possess tumor specific cytotoxicity; its expression induces apoptosis in human tumor and transformed cells but there is little or no cytotoxic effect in normal human cells or cell lines derived from different tissues including peripheral blood mononuclear cells, fibroblast and epithelial cells. Several studies have shown that the tumor specific killing of Apoptin correlates with its phosphorylation and its subcellular localization. In cancer cells, Apoptin is localized in the nucleus and is phosphorylated on threonine108 by an as yet unknown kinase, whereas in normal cells Apoptin is detected in the cytoplasm and is essentially unphosphorylated. We developed a lentiviral vector encoding a GFP-Apoptin fusion gene (LV-GFP-AP), which delivers the Apoptin gene efficiently to haematopoietic cells. Apoptin significantly and selectively killed a number of leukemia cell lines including K562, HL60, U937, KG1 and NB4. In particular, the dexamethasone resistant multiple myeloma cell line MM1.R and the dexamethasone sensitive cell line MM1.S were efficiently killed by Apoptin. In contrast normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, we showed that the dexamethasone resistant MM1.R cells were considerably more susceptible to Apoptin induced cell death than the parental matched MM1.S cells. This correlated with increased phosphorylation and activation of the Apoptin protein in MM1.R cells. Expression profiling of MM1.R and MM1.S cells identified a number of differentially expressed kinases. PKCb was over-expressed 9 fold in MM1.R cells and we showed, by immunoprecipitation and in vivo kinase studies, that this kinase was responsible for Apoptin phosphorylation. Analysis of the Apoptin amino acid sequence for potential phosphorylation sites indicated seven putative phosphorylation sites corresponding to the PKC kinase consensus motifs (S/TXK/R or S/TXXK/R). These sites included Thr-108, which has been previously shown to be phosphorylated in tumor cells, but not in normal cells. In vitro studies showed that recombinant Apoptin protein was phosphorylated by recombinant GST-PKCb protein at the Thr-108 site. Addition of a PKCb specific inhibitor resulted in diminished Apoptin phosphorylation whilst an unrelated inhibitor had no such effect. Furthermore, shRNA knockdown or drug mediated inhibition of PKCb in vivo significantly reduced Apoptin phosphorylation. Finally, we found that Apoptin mediated cell death proceeded via the up-regulation of PKCb, activation of caspase-9/3, cleavage of the PKCd catalytic domain and down-regulation of MERTK and AKT protein kinases. Collectively these results demonstrate a novel pathway for Apoptin activation involving PKCb and PKCd. Our results show that Apoptin is able to effectively eliminate multiple myeloma cells which have become resistant to dexamethasone. In addition, this study has led to the identification of tumor specific cellular targets such as PKCb, whose modulation by shRNAs and small molecule drugs can induce strong anti-myeloma effects. Importantly, the evidence from our data suggests that protein kinase C inhibitors may have an important therapeutic role in plasma cell neoplasia. Disclosures: No relevant conflicts of interest to declare.

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In vivo α-adrenergic responses and troponin I phosphorylation: anesthesia interactions

Journal of Applied Physiology ◽

10.1152/japplphysiol.00959.2004 ◽

2005 ◽

Vol 98 (4) ◽

pp. 1163-1170 ◽

Cited By ~ 8

Author(s):

Guy A. MacGowan ◽

Jennifer Rager ◽

Sanjeev G. Shroff ◽

Michael A. Mathier

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Transgenic Mice ◽

Troponin I ◽

Contractile Dysfunction ◽

Wild Type ◽

Adrenergic Stimulation ◽

Kinase C ◽

Significant Difference

The mechanisms by which α-adrenergic stimulation of the heart in vivo can cause contractile dysfunction are not well understood. We hypothesized that α-adrenergic-mediated contractile dysfunction is mediated through protein kinase C phosphorylation of troponin I, which in in vitro experiments has been shown to reduce actomyosin Mg-ATPase activity. We studied pressure-volume loops in transgenic mice expressing mutant troponin I lacking protein kinase C phosphorylation sites and hypothesized altered responses to phenylephrine. As anesthesia agents can produce markedly different effects on contractility, we studied two agents: avertin and α-chloralose-urethane. With α-chloralose-urethane, at baseline, there were no contractile abnormalities in the troponin I mutants. Phenylephrine produced a 50% reduction in end-systolic elastance in wild-type controls, although a 9% increase in troponin I mutants ( P < 0.05). Avertin was associated with reduced contractility compared with α-chloralose-urethane. Avertin anesthesia, at baseline, produced a reduction in end-systolic elastance by 31% in the troponin I mutants compared with wild-type ( P < 0.05), and this resulted in further marked systolic and diastolic dysfunction with phenylephrine in the troponin I mutants. Dobutamine produced no significant difference in the contractile phenotype of the transgenic mice with either anesthetic regimen. In conclusion, these data (α-chloralose-urethane) demonstrate that α-adrenergic-mediated force reduction is mediated through troponin I protein kinase C phosphorylation. β-Adrenergic responses are not mediated through this pathway. Altering the myofilament force-calcium relationship may result in in vivo increased sensitivity to negative inotropy. Thus choice of a negative inotropic anesthetic agent (avertin) with phenylephrine can lead to profound contractile dysfunction.

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Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light

Biochemical Journal ◽

10.1042/bj20030408 ◽

2003 ◽

Vol 374 (2) ◽

pp. 537-543 ◽

Cited By ~ 10

Author(s):

Ryan A. ADAMS ◽

Xinran LIU ◽

David S. WILLIAMS ◽

Alexandra C. NEWTON

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Outer Segment ◽

Phorbol Esters ◽

Phosphorylation Sites ◽

Rod Outer Segment ◽

Kinase C ◽

Rhodopsin Kinase

Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser334 and Ser338, are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser338 that is evenly distributed along the rod outer segment. Phosphorylation of Ser334 is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase−/− mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser334. In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser334, with no significant effect on the phosphorylation of Ser338. Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser334, which is regulated by protein kinase C.

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