The protein phosphatases involved in cellular regulation
Authors: TUNG H. Y. Lim , PELECH Steven , FISHER Michael J. , POGSON Christopher I. , COHEN Philip 
Published: March 2005

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.