Adenosine 5'-0-(3-thiotriphosphate) and CAMP-dependent protein kinase activity

Adenosine 5'-0-(3-thiotriphosphate) (ATPγS), analog of ATP, has been shown to be an effective substrate for various enzymes substituting for ATP, including some protein kinases (PK). Also, it has been reported that thiophosphorylated proteins are singularly resistant to dephosphorylating action of protein phosphatases. However, virtually no information is available concerning use of ATPγS as a substrate for cAMP-dependent protein kinase (cAMP-PK). The authors examined whether ATPγS would serve as a substrate for cAMP-PK activity contained in 27,000 x g supernatant of rat renal cortical homogenate. 32P-γ-ATP and ATPγ 35S were compared under the same conditions; kemptide or f2b histones served as a cAMP-PK substrate. Although 32P-ATP and ATPγ 35S were incorporated in the protein, only phosphorylation with 32P-γ-ATP, but not with ATPγ32S, was stimulated by cAMP in range 10-8 to 10-5M. Addition of unlabeled ATP to 32PγATP caused decreased 32P incorporation due to radiodilution. On the other hand, addition of unlabeled ATPγS had no such effect. Addition of unlabeled ATPγS (10-8 to 10-5M) did not influence specific activity of cAMP-PK or its stimulability by cAMP. Unlike in other enzymatic reaction, ATPγS cannot serve as a substrate or as a modulator for cAMP-dependent protein kinase