Abstract
Recently, we reported that pp60c-src kinase activity was present in adult bovine coronary arterial smooth muscle and showed that the activity of the enzyme in in vitro immunoprecipitation assays was stimulated 20-60-fold by ATP (Di Salvo, J., Gifford, D., and Kokkinakis, A. (1988) Biochem. Biophys. Res. Commun. 153, 388-394). In the present study, ATP-mediated stimulation of activity was also demonstrated in extracts from aortic vascular smooth muscle. In contrast, no stimulation was apparent in extracts from brain. Stimulation of activity in vascular preparations was also induced with beta,gamma-imidoadenosine 5'-triphosphate (AMP.PNP), a nonmetabolizable analog of ATP, and with several other polyphosphates including ADP and sodium pyrophosphate. No stimulation occurred in response to monophosphates such as AMP or KH2PO4. As expected, the specific activity of pp60c-src in brain extracts did not change when the amount of extracted protein included in immunoprecipitation mixtures was increased. Unexpectedly, however, the specific activity of the vascular enzyme decreased markedly as the amount of extracted protein subjected to immunoprecipitation was increased. Following stimulation of pp60c-src in vascular extracts with ATP, the enzyme behaved in a fashion similar to pp60c-src extracted from brain. That is, the enhanced specific activity of the stimulated vascular enzyme did not decrease with increasing amounts of extracted protein. Moreover, mixing experiments in which vascular smooth muscle extracts were added to brain extracts showed that the muscle extracts contained a factor which inhibited pp60c-src kinase activity. This inhibition was blocked when the mixed extracts were immunoprecipitated in the presence of ATP, or when inhibitory extract was treated with trypsin. Taken together, these data suggest that pp60c-src kinase activity in vascular tissue may be subject to a novel regulatory mechanism involving an inhibitory protein factor which can be nullified by polyphosphates.
Highlights
Several lines of evidence developed in this study suggest the interesting hypothesis that thekinase activity of pp60'"x in mammalian vascular smooth muscle may bemodulated by a polyphosphate-sensitive inhibitory factor, which isprobably protein in nature
Total activity which is measured in vascular extracts is either unchanged or decreased as the amount of extract analyzed is increased, whereas thetotal activity in brain extracts increases with the amount of extract assayed (Fig. 4)
Activity attained in a mixture of extracts from brain and atriumis comparable to thesum of the activities measured in the brain extract alone and in the atrialextract alone
Summary
Min and centrifuged for 30 min at 15,000 rpm (Sorvall, SS-34 rotor, 4 "C). Each supernatant was transferred to a precooled Eppendorf tube (1.5 ml), stored on ice for an additional 15 min, andcentrifuged for 5 min a t maximum speed in a Beckman Microfuge at 4 "C. Pp60""" kinase activityas a function of the amount of tissue protein (0.8-2.4 mg) included in immunoprecipitation mixtures containingextracts from bovine brain ( A ) ,coronary artery ( B ) ,and aortic muscularis (C).For each amount of protein tested, immunoprecipitates were prepared in the absence using a load of 0.8 mg of protein was 17 fmol of "P/min, whereas the activitywas about %fold higher (56 fmol of "P/ min) when 2.4 mg of extracted protein was included in the immunoprecipitation mixture Specific activities under these conditions were, respectively, 21.3 and 23.3 fmol of "P/min/. Stimulation of activity in aortic extracts increased progressively with low to moderate protein loads (0.4-1.6 mg, Fig. 4C), stimulation with 1 mM ATP was no longer present at the highest protein load (2.4 mg), and it was blunted in response to 5 mM ATP These data suggested that vascular extracts contained an inhibitor ofpp60""'" kinase whichcould be blocked by treatment with polyphosphates.
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