SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1

A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2890%2980245-E

Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants.

Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase

The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling.

Publisher Summary This chapter describes the purification of two forms of protein phosphatase- 1 (PP-I 1 and PP-l G ), and three forms of protein phosphatase-2A (PP-2A 0 , PP-2A 1 , and PP-2A 2 ) from skeletal muscle. Procedures for isolating the free catalytic subunits (termed PP-l c and PP-2A c ) are documented and the structures of these enzymes are summarized in the chapter. The free catalytic subunits are isolated by a modification of the procedure of Lee and co-workers. Skeletal muscle extracts prepared from 3000 g of muscle are adjusted to pH 7.2 with 10 M ammonium hydroxide, and 350 g of solid ammonium sulfate are added to each liter of solution to bring the degree of saturation to 55%. After standing for 30 min the suspension is centrifuged for 40 min at 4200 g and the supernatant discarded. The PP-2A c from poly(L-lysine)-Sepharose is purified in an identical manner to PP-I c .

Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2882%2980760-6

Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism

Homogenous preparations of the catalytic subunit of protein phosphatase-1 and inhibitor-2 can be combined to produce an inactive enzyme that consists of a 1:1 complex between these two proteins. This species is indistinguishable from the Mg-ATP-dependent protein phosphatase in that preincubation with glycogen synthase kinase-3 and Mg-ATP is required to generate activity. Activation results from the phosphorylation of inhibitor-2. The molar concentrations of protein phosphatase-1 and inhibitor-2 in rabbit skeletal muscle (0.25–0.5 μM) are similar.



Incubation of the reconstituted Mg-ATP-dependent protein phosphatase with chymotrypsin is accompanied by limited proteolysis of inhibitor-2 and the loss of its phosphorylation site(s). This species can be activated by glycogen synthase kinase-3 and Mg-ATP provided that inhibitor-2 is added. This exogenous inhibitor-2 appears to displace the fragments of inhibitor-2 from the enzyme that were generated by chymotryptic digestion. These experiments may explain the report [Yang, S. D., Vandenheede, J. R. and Merlevede, W. (1981) J. Biol. Chem. 256, 10231–10234] that inhibitor-2 can function as an ‘activator’ as well as an inhibitor of the Mg-ATP-dependent protein phosphatase.



Incubation of the catalytic subunit of protein phosphatase-1 with sodium fluoride or sodium pyrophosphate converted the enzyme to an inactive from that could be partially reactivated by manganese ions, but not by glycogen synthase kinase-3 and Mg-ATP. Conversely, the reconstituted Mg-ATP-dependent protein phosphatase could only be activated by glycogen synthase kinase-3 and Mg-ATP, and not by manganese ions. It is concluded that the conversion of protein phosphatase-1 to a manganese-ion dependent form is a quite separate phenomenon from the formation of the Mg-ATP-dependent protein phosphatase.



Inhibitor-2 can inactivate protein phosphatase-1 by a second mechanism that is not reversed by preincubation with glycogen synthase kinase-3 and Mg-ATP. This occurs at higher concentrations of inhibitor-2 than those required to form the Mg-ATP-dependent protein phosphatase, and appears to result from the binding of inhibitor-2 to a distinct site on the enzyme.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07485.x

Therese J. Resink

Papers

Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle.

Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism

Characterisation of a reconstituted Mg-ATP-dependent protein phosphatase.

Amino acid sequence at the site on protein phosphatase inhibitor-2, phosphorylated by glycogen synthase kinase-3

Protein phosphorylation and the neural and hormonal control of enzyme activity1