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First published online 23 March 2004
doi: 10.1242/jcs.01044

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Myofilament anchoring of protein kinase C-epsilon in cardiac myocytes

¹ Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431, USA
² Department of Physiology, University of Wisconsin, Madison, WI 53706, USA

View larger version (17K): [in a new window]   Fig. 1. -PKC western blot standard curve. Purified -PKC was subjected to SDS-PAGE and immunoblotted as described in Materials and Methods. (A) Bands were visualized by ECL and quantified by densitometry. (B) Integrated optical densities (OD) were plotted as a function of -PKC mass. To ensure linearity of -PKC binding in all subsequent experiments, only OD values below 13 were used for quantification.

View larger version (15K): [in a new window]   Fig. 2. Binding of -PKC to cardiac myofilaments. (A) Western blot of myofilament pellets showing binding of purified -PKC in absence or presence of 50 µM arachidonic acid (AA). -PKC concentrations (in µM): lane 1, 0; lane 2, 0.1; lane 3, 0.2; lane 4, 0.5; lane 5, 1. (B) Summary of -PKC binding to myofilaments. The solid circles (in the presence of AA) represents a fit to B/Bmax=[-PKC]/([-PKC] + EC50), with EC50=86 nM and Bmax=15 pmol (or 75 pmol/mg protein). The stoichiometry of binding was 15 pmols -PKC bound per 2 nmoles actin monomers or a 1/130 ratio. The open circles represent a linear regression of -PKC binding in the absence of AA.

View larger version (112K): [in a new window]   Fig. 3. Phase contrast (A) and fluorescent (B) images of myofilament associated -PKC. -PKC is activated with 50 µM arachidonic acid (AA) and the activated -PKC is associated with myofilaments near Z-lines as indicated by arrows in the overlay image (C). A confocal image of a skinned myocyte decorated with -PKC, an anti--PKC primary antibody and Alexa 488 secondary antibody (D) Magnification x1000.

View larger version (27K): [in a new window]   Fig. 4. Isoform specificity of PKC binding to cardiac myofibrils. (A) Western blots of myofibril pellets after incubation with -PKC, -PKC or -PKC. Below each lane is indicated the PKC activators used to stimulate binding. na, No activators. AA, 50 µM arachidonic acid. AA + DAG, 50 µM AA plus 25 µM dioctanoylglycerol. M is -PKC gel/blot marker, and M is -PKC gel/blot marker.

View larger version (16K): [in a new window]   Fig. 5. Inhibition of -PKC binding to cardiac myofibrils by synthetic peptides. (A) A Western blot shows -PKC in the myofibril pellet is reduced in a concentration-dependent manner by the octapeptide, EAVSLKPT. The F-actin binding hexapeptide LKKQET had no effect of -PKC binding to myofibrils over the same concentration range. -PKC concentration was 100 nM and peptide concentrations in µM are given above each lane. (B) Summary of peptide inhibition of -PKC binding to cardiac myofibrils. Bars represent mean±s.e.m. of five experiments.

View larger version (10K): [in a new window]   Fig. 6. Properties of -PKC binding to isolated F-actin. (A) Summary of -PKC binding to F-actin in the absence and presence of 50 µM arachidonic acid (AA). The line with solid circles (in the presence of AA) represents a fit to: B/Bmax=[-PKC]/([-PKC] + EC50), with EC50=110 nM and Bmax=18 pmol (or 180 pmol/mg protein). The stoichiometry of binding was 18 pmols -PKC bound per 2 nmoles actin monomers or a 1/110 ratio. Open circles represent -PKC with F-actin in the absence of AA. (B) Summary of inhibition of -PKC binding to F-actin by synthetic peptides. Data is represented as mean±s.e.m. from five experiments.

View larger version (63K): [in a new window]   Fig. 7. Absence of Cypher-1 in skinned cardiac myofibrils. Left Panel: Coomassie Blue-stained SDS-PAGE gel of intact (in) and Triton X-100 skinned (sk) rat ventricular myocytes. Protein loads were 20 µg (left two lanes) and 10 µg (right two lanes). Right panel: Western blot of same samples as in left panel. Arrowheads mark the mobility of Cypher-1.