First published online 26 July 2005
doi: 10.1242/jcs.02493
Journal of Cell Science 118, 3595-3605 (2005)
Published by The Company of Biologists 2005
MARCKS is a major PKC-dependent regulator of calmodulin targeting in smooth muscle
Cynthia Gallant1,
Jae Young You2,
Yasuharu Sasaki3,
Zenon Grabarek1 and
Kathleen G. Morgan1,4,*
1 Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA
2 Wellesley College, 106 Central Street, Wellesley, MA 02481, USA
3 Department Pharmacology and Pharmaceutical Science, Kitasato University, Tokyo 108-8641, Japan
4 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
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Fig. 1. CaM distribution is heterogeneous and agonist-dependent. (A) A single, live, freshly dissociated smooth-muscle cell loaded with AF-CaM. The arrowhead denotes the position of the nucleus. (B) CaM immunoblot of portal-vein homogenate demonstrating specificity of the antibody used for imaging. (C) Endogenous CaM distribution monitored by indirect immunofluorescence. (top) Control, unstimulated cell. (Inset) Digitally expanded image to illustrate interplaque distance. (middle) 10 minutes, 51 mM KCl PSS. (bottom) 20 minutes, 10 µM DPBA. Arrows point to the location of filamentous structures. Arrowheads denote the position of the nucleus. (D) Quantitation of endogenous CaM distribution. Bars represent the average ratios of surface to core fluorescence ratios (s/c) of fluorescence from 9-21 cells under control conditions or 10 minutes after 10 µM DPBA or 51 mM KCl PSS. (inset) A diagrammatic example of the output from a single line scan in the region of a cell marked with the white line. ***, P<0.001 compared with control; ##, P<0.01 compared with DPBA treatment.
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Fig. 2. Abundance of MARCKS and CaM in smooth muscle. (A) MARCKS immunoblot of whole-cell homogenate of portal vein. (B) Quantitation of portal-vein MARCKS immunoblot, protein matched for total cellular protein compared with a standard curve generated for calf-forebrain homogenates. (C) CaM immunoblot of whole-cell homogenate of portal vein. (D) Quantitation of portal-vein CaM immunoblots protein matched for total cellular protein compared with a standard curve generated by calf-forebrain homogenates.
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Fig. 3. MARCKS colocalizes with CaM and vinculin in adhesion plaques of unstimulated cells. (A) Centre confocal section of one freshly dissociated portal vein cell labelled for both MARCKS and CaM. Arrowheads denote the position of the nucleus. Insets show digitally expanded views of MARCKS and CaM staining. (B) A grazing surface optical section of one freshly dissociated portal-vein cell labelled for both MARCKS and CaM. White arrows indicate the specific repeating membrane structures. (C) A surface confocal section of one cell labelled for both MARCKS and vinculin. Composite images show overlap in yellow.
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Fig. 4. CaM interacts with MARCKS in vitro and in vivo in a Ca2+-dependent manner. (A, left) Total protein on PVDF membrane stained with Naphthol Blue Black before exposure to overlay buffer. (A, right) A parallel lane immunostained to locate MARCKS. (A, middle) CaM overlay. The lanes were treated as follows: 1, no Ca2+ was added to the overlay buffer and 10 mM EGTA was included; 2, overlay buffer contained 2.5 mM CaCl2. (B) Two freshly dissociated cells labelled for both CaM and MARCKS. (top) Cell fixed in PSS containing 1 mM Ca2+. (bottom) Cell fixed in Ca2+-free PSS containing 10 mM EGTA. Arrowheads denote the position of the nucleus.
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Fig. 5. CaM and MARCKS co-translocate in response to depolarization. (A) Centre sections of an unstimulated cell (top), a cell in 51 mM KCl PSS for 2.5 minutes (middle) and a cell in 51 mM KCl PSS for 10 minutes (bottom), all labelled for both CaM and MARCKS. Arrowheads denote the positions of the nuclei. (B) Line-scan analysis of CaM translocation in response to KCl PSS. The mean surface:core ratio as a percentage of the average resting ratio for 9-22 cells is shown versus time. (C) Line-scan analysis of MARCKS translocation in response to KCl PSS.
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Fig. 6. CaM and MARCKS translocate with different time courses in response to the phorbol ester DPBA. (A) Centre confocal sections of unstimulated or DPBA-stimulated cells labelled for both CaM and MARCKS. Arrowheads denote the positions of the nuclei. (B) Line-scan analysis of CaM translocation in response to 10 µM DPBA. The mean ratio of surface to core fluorescence of 12 to 24 cells is shown against time. (C) Line-scan analysis of MARCKS translocation in response to 10 µM DPBA. The mean surface to core fluorescence ratios for 19-26 cells are shown against time.
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Fig. 7. Stimulus-specific immunoprecipitation and photoaffinity cross-linking of MARCKS and MARCKS ED peptide with CaM. (A) Immunoprecipitation of portal-vein homogenates with an anti-CaM antibody followed by immunoblot with an anti MARCKS antibody (left) or anti-CaM antibody (right) after exposure of muscles to: no stimulus; 2.5 minutes KCl; or 2.5 minutes DPBA. A typical western blot is shown above the graph of mean±s.e. densitometry for repeated experiments. *, P<0.05; **, P<0.01 compared with unstimulated cells; ###, P<0.001 compared with KCl treatment. n=4. (B) Coomassie-stained SDS-PAGE gel of CaM mixed with ED-BPM peptide and exposed to no light (lane 1), UV light in the absence of Ca (lane 2) and UV light in the presence of Ca2+ (lane 3). (C) CaM immunoblot of portal-vein homogenates after muscles were chemically loaded with ED-BPM peptide and exposed to UV light in the presence of no stimulus (lane 2), 51 mM KCl PSS (lane 3) or DPBA (lane 4). Also, as a control, a mock preparation not loaded with peptide but exposed to UV light is shown (lane 1). (D) Average densitometry of the upper band in three experiments performed as described in C. #, P<0.05; ###, P<0.001 compared with unstimulated cells; ***, P<0.001 compared with DPBA treatment.
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Fig. 8. Time course of MARCKS phosphorylation in the presence of DPBA. (A) Domain map for human MARCKS sequence showing the N-terminal myristoylation site and the central effector domain (ED) or PhosphoSite Domain (PSD). Based on data reviewed in Blackshear (Blackshear, 1993 ). (B) Typical blots for DPBA-mediated Ser159 MARCKS or Ser159/163 MARCKS phosphorylation with time after addition of the phorbol ester DPBA. (C) Average densitometry of four to six pSer159 MARCKS immunoblots. (D) Average densitometry of five to seven pSer159/163 MARCKS immunoblots.
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Fig. 9. CaM translocation is blocked by a nonphosphorylatable MARCKS ED decoy peptide. (A) Confocal images of distribution of fluorescent ED4A or ED4Ax in freshly dissociated portal-vein smooth-muscle cells (left) and quantitation of average surface:core fluorescence ratios from 9-11 cells (right). Arrowheads denote the position of the nucleus. Where no arrowhead is shown, the nucleus is beyond the field of view of the image shown. (B, left) Confocal images of cells fixed and immunostained to locate CaM after loading with ED4A or ED4AX either at rest or after stimulation with the phorbol ester DPBA. (B, right) Average surface:core fluorescence ratios from 9-11 cells. Arrowheads denote the positions of the nuclei. Where no arrowhead is shown, the nucleus is beyond the field of view of the image shown. (C) Average densitometry of immunoblots for Ser159/163 MARCKS phosphorylation for unstimulated tissues or tissues stimulated with DPBA and mock loaded or loaded with ED4A or ED4AX (n=4). *, P<0.05 compared with the mock-loaded control; #, P<0.05 compared with ED4AX.
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© The Company of Biologists Ltd 2005
