Smooth muscle archvillin is an ERK scaffolding protein.
ERK influences a number of pathways in all cells, but how ERK activities are segregated between different pathways has not been entirely clear. Using immunoprecipitation and pulldown experiments with domain-specific recombinant fragments, we show that smooth muscle archvillin (SmAV) binds ERK and members of the ERK signaling cascade in a domain-specific, stimulus-dependent, and pathway-specific manner. MEK binds specifically to the first 445 residues of SmAV. B-Raf, an upstream regulator of MEK, constitutively interacts with residues 1-445 and 446-1250. Both ERK and 14-3-3 bind to both fragments, but in a stimulus-specific manner. Phosphorylated ERK is associated only with residues 1-445. An ERK phosphorylation site was determined by mass spectrometry to reside at Ser132. A phospho-antibody raised to this site shows that the site is phosphorylated during alpha-agonist-mediated ERK activation in smooth muscle tissue. Phosphorylation of SmAV by ERK decreases the association of phospho-ERK with SmAV. These results, combined with previous observations, indicate that SmAV serves as a new ERK scaffolding protein and provide a mechanism for regulation of ERK binding, activation, and release from the signaling complex.
Gangopadhyay
Samudra S
SS
Boston University, Boston, Massachusetts 02215, USA.
Kengni
Edouard
E
Appel
Sarah
S
Gallant
Cynthia
C
Kim
Hak Rim
HR
Leavis
Paul
P
DeGnore
Jon
J
Morgan
Kathleen G
KG
D048948:14-3-3 Proteins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D005289:Ferrets; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism; D015398:Signal Transduction
D048948:14-3-3 Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases