In vivo screening for substrates of protein kinase A using a combination of proteomic approaches and pharmacological modulation of kinase activity.
Protein kinase A (PKA) is a serine/threonine kinase whose activity depends on the levels of cyclic AMP (cAMP). PKA plays essential roles in numerous cell types such as myocytes and neurons. Numerous substrate screens have been attempted to clarify the entire scope of the PKA signaling cascade, but it is still underway. Here, we performed a comprehensive screen that consisted of immunoprecipitation and mass spectrometry, with a focus on the identification of PKA substrates. The lysate of HeLa cells treated with Forskolin (FSK)/3-isobutyl methyl xanthine (IBMX) and/or H-89 was subjected to immunoprecipitation using anti-phospho-PKA substrate antibody. The identity of the phosophoproteins and phosphorylation sites in the precipitants was determined using liquid chromatography tandem mass spectrometry (LC/MS/MS). We obtained 112 proteins as candidate substrates and 65 candidate sites overall. Among the candidate substrates, Rho-kinase/ROCK2 was confirmed to be a novel substrate of PKA both in vitro and in vivo. In addition to Rho-kinase, we found more than a hundred of novel candidate substrates of PKA using this screen, and these discoveries provide us with new insights into PKA signaling.
Department of Cell Pharmacology, Nagoya University, Graduate School of Medicine.
Tomonari
T
Hamaguchi
Shinichi
S
Nakamuta
Yasuhiro
Y
Funahashi
Tetsuya
T
Takano
Tomoki
T
Nishioka
Md Hasanuzzaman
MH
Shohag
Yoshimitsu
Y
Yura
Kozo
K
Kaibuchi
Mutsuki
M
Amano
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007546:Isoquinolines / Q000494:pharmacology; D013058:Mass Spectrometry; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D040901:Proteomics; D013449:Sulfonamides / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000737:chemistry / Q000378:metabolism
D007546:Isoquinolines; D013449:Sulfonamides; D005576:Colforsin; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D015056:1-Methyl-3-isobutylxanthine