Protein kinase activity of phosphoinositide 3-kinase regulates beta-adrenergic receptor endocytosis.
Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent beta-adrenergic receptor (betaAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent betaAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) complements the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective betaAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions if tropomyosin resulted in complete inhibition of betaAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in betaAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.
Naga Prasad
Sathyamangla V
SV
Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Jayatilleke
Arundathi
A
Madamanchi
Aasakiran
A
Rockman
Howard A
HA
D000199:Actins / Q000378:metabolism; D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D019390:Arrestins / Q000378:metabolism; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009154:Mutation / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism; D000077191:Wortmannin; D051552:beta-Adrenergic Receptor Kinases; D000071557:beta-Arrestins
D000199:Actins; D033962:Adaptor Protein Complex 2; D000730:Androstadienes; D019390:Arrestins; D006657:Histones; D018129:Phosphatidylinositol Phosphates; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D011943:Receptors, Adrenergic, beta; D018343:Receptors, Adrenergic, beta-2; D014168:Transferrin; D014335:Tropomyosin; D000071557:beta-Arrestins; C060980:phosphatidylinositol 3,4-diphosphate; D012694:Serine; D011494:Protein Kinases; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D000077191:Wortmannin