Identification of filamin C as a new physiological substrate of PKBalpha using KESTREL.
We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by PKBa (protein kinase Ba), but not by SGK1 (serum- and glucocorticoid-induced kinase 1), and identified it as the cytoskeletal protein FLNc (filamin C). PKBa phosphorylated FLNc at Ser2213 in vitro, which lies in an insert not present in the FLNa and FLNb isoforms. Ser2213 became phosphorylated when C2C12 myoblasts were stimulated with insulin or epidermal growth factor, and phosphorylation was prevented by low concentrations of wortmannin, at which it is a relatively specific inhibitor of phosphoinositide 3-kinase. PD 184352 [an inhibitor of the classical MAPK (mitogen-activated protein kinase) cascade] and/or rapamycin [an inhibitor of mTOR (mammalian target of rapamycin)] had no effect. Insulin also induced the phosphorylation of FLNc at Ser2213 in cardiac muscle in vivo, but not in cardiac muscle that does not express PDK1 (3-phosphoinositide-dependent kinase 1), the upstream activator of PKB. These results identify the muscle-specific isoform FLNc as a new physiological substrate for PKB.
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK. j.t.c.murray@dundee.ac.uk
James T
JT
Murray
David G
DG
Campbell
Mark
M
Peggie
Alfonso
A
Mora
Mora
M
Alfonso
Philip
P
Cohen
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity; D001549:Benzamides / Q000494:pharmacology; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D003285:Contractile Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D064448:Filamins; D006801:Humans; D017874:Immediate-Early Proteins; D007328:Insulin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D013379:Substrate Specificity; D000077191:Wortmannin; D000099184:Serum-Glucocorticoid Regulated Kinases
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000730:Androstadienes; D001549:Benzamides; D002457:Cell Extracts; D003285:Contractile Proteins; D004815:Epidermal Growth Factor; D064448:Filamins; D017874:Immediate-Early Proteins; D007328:Insulin; D007527:Isoenzymes; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D000077191:Wortmannin; D000099184:Serum-Glucocorticoid Regulated Kinases; C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; C578662:FLNB protein, human; C578661:FLNC protein, human; C578562:PDPK1 protein, human; C578564:Pdpk1 protein, mouse