Heat-stable regulatory factors are associated with polycation-modulable phosphates

Abstract Several protein phosphatases which we designated PCM-I, PCM-II and PCM-II d 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 lyine-rich histone-H 1 . 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-II d . The enzymes appear similar in that low concentrations of polylysine (0.03–0.13 μ m ) 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-II d 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 μ m ), but no stimulation is seen with PCM-II d . In addition, earlier studies with PCM-I showed that, unlike either PCM-II or PCM-II d 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°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. Interaction between the regulatory proteins and polycationic effectors could result in conformational changes such that phosphatase activity expressed against one substrate is enhanced, while activity expressed against a different substrate is reduced. Within this framework, the appearance of multiple forms of PCM-phosphatase, as well as the specific response of each form to polycationic modulation of relative substrate specificity, may be attributable to selective loss or acquisition of one or more regulatory proteins. Although further studies are required to test this hypothesis it provides a useful working model for interpreting some of our data and for projecting future experiments. Conceivably, modulation of PCM-phosphatase activity and relative substrate specificity by polycationic effectors and/or specific regulatory proteins may be an important mechanism for coordinating different cellular mechanisms such as arterial metabolism and contractility.