Smooth muscle archvillin: a novel regulator of signaling and contractility in vascular smooth muscle.
The mechanisms by which protein kinase C (PKC) and extracellular-signal-regulated kinases (ERK1/2) govern smooth-muscle contractility remain unclear. Calponin (CaP), an actin-binding protein and PKC substrate, mediates signaling through ERK1/2. We report here that CaP sequences containing the CaP homology (CH) domain bind to the C-terminal 251 amino acids of smooth-muscle archvillin (SmAV), a new splice variant of supervillin, which is a known actin- and myosin-II-binding protein. The CaP-SmAV interaction is demonstrated by reciprocal yeast two-hybrid and blot-overlay assays and by colocalization in COS-7 cells. In differentiated smooth muscle, endogenous SmAV and CaP co-fractionate and co-translocate to the cell cortex after stimulation by agonist. Antisense knockdown of SmAV in tissue inhibits both the activation of ERK1/2 and contractions stimulated by either agonist or PKC activation. This ERK1/2 signaling and contractile defect is similar to that observed in CaP knockdown experiments. In A7r5 smooth-muscle cells, PKC activation by phorbol esters induces the reorganization of endogenous, membrane-localized SmAV and microfilament-associated CaP into podosome-like structures that also contain F-actin, nonmuscle myosin IIB and ERK1/2. These results indicate that SmAV contributes to the regulation of contractility through a CaP-mediated signaling pathway, involving PKC activation and phosphorylation of ERK1/2.
Gangopadhyay
Samudra S
SS
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA.
Takizawa
Norio
N
Gallant
Cynthia
C
Barber
Amy L
AL
Je
Hyun-Dong
HD
Smith
Tara C
TC
Luna
Elizabeth J
EJ
Morgan
Kathleen G
KG
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism; D015153:Blotting, Western; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D005289:Ferrets; D005982:Glutathione Transferase / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D008840:Microfilament Proteins / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000096985:Calponins
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D005982:Glutathione Transferase; D008565:Membrane Proteins; D008840:Microfilament Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016376:Oligonucleotides, Antisense; D011493:Protein Kinase C; D011994:Recombinant Proteins; D000096985:Calponins; C491615:Svil protein, mouse