The protein phosphatases involved in cellular regulation

The effects of polyamines on the oligomeric forms of protein phosphatase-1 (1G), protein phosphatase-2A (2A0, 2A1 and 2A2) and their free catalytic subunits (1C and 2AC) has been studied using homogeneous enzymes isolated from rabbit skeletal muscle. Spermine increased the activity of protein phosphatase-2A towards eight of nine substrates tested. Half-maximal activation was observed at 0.2 mM with optimal effects at 1–2 mM. Above 2 mM, spermine became inhibitory. The most impressive activation of protein phosphatase-2A was obtained with glycogen synthase, especially when phosphorylated at sites-3 (8–15-fold with protein phosphatase-2A1) and phenylalanine hydroxylase (6–7-fold with protein phosphatase-2A1) as substrates. Activation of protein phosphatases 2A0, 2A1 and 2A2 was greater than that observed with 2AC. Spermine was a more potent activator than spermidine, while putrescine had only a small effect. Qualitatively similar results were obtained with five other substrates, although maximal activation was much less (1.3–3-fold with protein phosphatase-2A1). The rate of dephosphorylation of glycogen phosphorylase was decreased by spermine, inhibition being more pronounced with protein phosphatase-2AC than with 2A0, 2A1 and 2A2. Spermine (I50= 0.1 mM with protein phosphatase-2AC) was a more potent inhibitor than spermidine (I50= 0.9 mM) or putrescine (I50= 8 mM). Partially purified preparations of protein phosphatases-2A0, 2A1 and 2A2 from from rat liver were affected by spermine in a similar manner to the homogeneous enzymes from rabbit skeletal muscle. Spermine did not activate protein phosphatase-1 to the same extent as protein phosphatase-2A. Greatest stimulation (2.5-fold) was again observed with glycogen synthase labelled in sites-3, with half-maximal activation at 0.2 mM and optimal effects at 1–2 mM spermine. Spermine was a much more effective stimulator than spermidine, while putrescine was ineffective. Very similar results were obtained with protein phosphatases 1G and 1C. With four other substrates maximal activation by spermine was < 1.5-fold, while the dephosphorylation of glycogen synthase (labelled in site-2), phosphorylase kinase, pyruvate kinase and glycogen phosphorylase were inhibited. Spermine (I50= 0.04 mM) was a more potent inhibitor of the dephosphorylation of glycogen phosphorylase than spermidine (I50= 0.9 mM) or putrescine (I50= 9 mM). There was some resemblance between the effects of Mn2+ and spermine on the activity of protein phosphatase-2A, although with the substrates inhibitor-1 and phenylalanine hydroxylase, activation by Mn2+ was greater than that observed with spermine, while with glycogen synthase the converse was true. Mn2+ could not mimic the activating effect of spermine on dephosphorylation of glycogen synthase (labelled in sites-3) by protein phosphatase 1. By contrast, spermine hardly affected the dephosphorylation of inhibitor-1 by phosphatase-1, a reaction that was almost completely dependent on Mn2+. Physiological concentrations of Mg2+ (1.0 mM) neither mimicked, nor influenced, the response to spermine with either protein phosphatase-1 or 2A. The effects of spermine appear to result from interaction of the polyamine with both the protein phosphatases and their substrates. The possibility that activation of protein phosphatase-1 and/or 2A by spermine underlies the relatively specific dephosphorylation of sites-3 of glycogen synthase by insulin is discussed.