ATP- and polyphosphate-mediated stimulation of pp60c-src kinase activity in extracts from vascular smooth muscle.

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.

pp60c-src kinase activity in bovine coronary extracts is stimulated by ATP.

pp60c-src kinase is believed to participate in regulating key cellular mechanisms including signal transduction and differentiation of smooth muscle during early embryogenesis. In this study, pp60c-src kinase activity was demonstrated in extracts from adult bovine coronary arterial smooth muscle. Activity, reflected by autophosphorylation of pp60c-src, phosphorylation of exogenous substrates, and phosphorylation of several endogenous substrates, was enhanced about 2 fold when added Mg2+ was replaced by Mn2+. Unexpectedly, activity was dramatically stimulated 20-50 fold by prior incubation with ATP. Such stimulation appears to be mediated through a novel mechanism which is independent of ATP-induced phosphorylation of reaction components. These new observations strongly suggest that a unique mechanism exists for regulation of coronary arterial pp60c-src kinase activity. Conceivably, this mechanism may serve important roles in modulating signal transduction and contractility of vascular smooth muscle.

A multisubstrate Ca2+ and cyclic nucleotide independent kinase from vascular smooth muscle: modulation of activity by polycations.

A multisubstrate Ca2+ and cyclic nucleotide independent kinase (Mr = 47,000) was purified from bovine aortic smooth muscle. Phosphorylation of glycogen synthase by this enzyme was polycation modulable. Low concentrations of polylysine (0.04-0.16 microM) stimulated phosphorylation 2-7 fold, whereas higher concentrations suppressed phosphorylation. Glycogen synthase converted to its glucose 6-PO4 dependent form following phosphorylation in either the presence (7 mol 32P/mol synthase) or absence (4 mol 32P/mol synthase) of polylysine: extent of conversion correlated to extent of phosphorylation. Seven of 14 potential substrates tested were phosphorylated: kinase activity was greatest for phosvitin followed by casein, the receptor protein from type 2 cAMP-kinase, histone H2b, phosphorylase kinase, glycogen synthase, and myocardial myosin light chains. Phosphorylation of phosvitin or synthase was inhibited by heparin (1/2 maximally by 0.5 microgram/ml without salt and 37 micrograms/ml with 150 mM NaCl). The results suggest that the enzyme may participate in regulating arterial glycogen metabolism and that such regulation may be modulated by polycationic and polyanionic effectors.

A new heat-stable regulatory factor is associated with aortic polycation-modulated (PCM)-phosphatase.

An aortic phosphatase which dephosphorylates several proteins including phosphorylase a and the 20-kDa myosin light chains is subject to modulation in vitro by polycationic effectors such as lysine-rich histone-H1 and polylysine. This study was based on the hypothesis that polycationic modulation of expressed enzymic activity involves interactions between the effectors and a regulatory site associated with the polycation-modulated (PCM)-phosphatase. Basal PCM-phosphatase activity expressed against myocardial myosin light chains (MLC, 1258 nmole/min/mg) was about eightfold greater than activity expressed against phosphorylase a (149 nmole/min/mg). However, dephosphorylation of phosphorylase a was stimulated four- to sevenfold by low concentrations of polylysine (Mr = 13,000; 0.01-0.1 microM), whereas MLC phosphatase activity was virtually abolished. Higher concentrations of polylysine inhibited dephosphorylation of either substrate. Interestingly, a heat-stable fraction prepared from the PCM-phosphatase reversed the stimulatory effect of polylysine on phosphorylase phosphatase activity and the inhibitory effect on dephosphorylation of MLC. No reversal of the modulatory effects of polylysine occurred when protein phosphatase inhibitor 1 or inhibitor 2 was substituted for the heat-stable factor derived from the PCM-phosphatase. Sucrose density centrifugation of the enzyme yielded a single peak (Mr = 63,000) exhibiting polycation-modulated activity against phosphorylase a and MLC. Moreover, heating each of the gradient fractions showed the presence of a heat-stable factor which reversed the modulatory effects of polylysine on dephosphorylation of either phosphorylase a or MLC. These results show that a specific heat-stable factor, which differs from both inhibitor 1 and 2, is associated with the PCM-phosphatase. The results suggest that polycationic modulation of expressed PCM-phosphatase activity may involve interactions between the polycationic effector and the enzyme-associated regulatory factor.

Heat-stable regulatory factors are associated with polycation-modulable phosphatases.

Several protein phosphatases which we designated PCM-I, PCM-II and PCM-IId were identified in preparations from aortic smooth muscle. A unique feature of these enzymes is that phosphatase activities expressed against different substrates are subject to modulation by polycationic effectors such as polylysine and lysine-rich histone-H1. They can be distinguished from each other by virtue of significant quantitative differences regarding (a) relative substrate specificities exhibited against phosphorylated myosin light chains, phosphorylase, and inhibitor-1, (b) apparent molecular weights as determined by sucrose density centrifugation, and (c) differential susceptibility to polylysine-mediated modulation of phosphatase activities. Surprisingly, gel filtration of the very same PCM-phosphatase preparation yields either of two apparently different enzymes: namely PCM-II, or PCM-IId. The enzymes appear similar in that low concentrations of polylysine (0.03-0.13 microM) inhibit dephosphorylation of light chains by either enzyme and high concentrations inhibit dephosphorylation of phosphorylase a. However, PCM-II exhibits higher basal phosphatase activity against myosin light chains (480 U/mg) than against phosphorylase a (175 U/mg). In contrast, PCM-IId is more effective in dephosphorylating phosphorylase a (180 U/mg) than in dephosphorylating the light chains (88 U/mg). Moreover, phosphorylase phosphatase activity of PCM-II is biphasically stimulable by low concentrations of polylysine (0.01-0.5 microM), but no stimulation is seen with PCM-IId. In addition, earlier studies with PCM-I showed that, unlike either PCM-II or PCM-IId polylysine biphasically stimulated the dephosphorylation of both phosphorylase a and the myosin light chains. Nevertheless, in spite of these obvious differences between the enzymes other data suggests that the PCM-phosphatases may be related to each other. Incubation of PCM-II at 90 degrees C for 10 min apparently releases heat-stable regulatory proteins which reverse the modulatory effects of polylysine on light chain and phosphorylase phosphatase activities expressed by either PCM-II or PCM-I. Similarly, heat-stable proteins released from PCM-I also reverse polylysine-mediated modulation of both PCM-I and PCM-II. These findings are consistent with a working hypothesis suggesting that PCM-phosphatase may consist of a modulatory domain containing several regulatory proteins and a catalytic domain.(ABSTRACT TRUNCATED AT 400 WORDS)