The protein phosphatases involved in cellular regulation: Influence of polyamines on the activities of protein phosphatase‐1 and protein phosphatase‐2A

The effects of polyamines on the oligomeric forms of protein phosphatase‐1 (1_G), protein phosphatase‐2A (2A₀, 2A₁ and 2A₂) and their free catalytic subunits (1_C and 2A_C) 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 2A_C. 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‐2A_C than with 2A₀, 2A₁ and 2A₂. Spermine (I₅₀= 0.1 mM with protein phosphatase‐2A_C) 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 1_G and 1_C. 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.