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Rac GTPase signaling through the PP5 protein phosphatase.

ABSTRACT: We have investigated the Rac-dependent mechanism of KCNH2 channel stimulation by thyroid hormone in a rat pituitary cell line, GH(4)C(1), with the patch-clamp technique. Here we present physiological evidence for the protein serine/threonine phosphatase, PP5, as an effector of Rac GTPase signaling. We also propose and test a specific molecular mechanism for PP5 stimulation by Rac-GTP. Inhibition of PP5 with the microbial toxin, okadaic acid, blocked channel stimulation by thyroid hormone and by Rac, but signaling was restored by expression of a toxin-insensitive mutant of PP5, Y451A, which we engineered. PP5 is unique among protein phosphatases in that it contains an N-terminal regulatory domain with three tetratricopeptide repeats (TPR) that inhibit its activity. Expression of the TPR domain coupled to GFP blocked channel stimulation by the thyroid hormone. We also show that the published structures of the PP5 TPR domain and the TPR domain of p67, the Rac-binding subunit of NADPH oxidase, superimpose over 92 alpha carbons. Mutation of the PP5 TPR domain at two predicted contact points with Rac-GTP prevents the TPR domain from functioning as a dominant negative and blocks the ability of Y451A to rescue signaling in the presence of okadaic acid. PP5 stimulation by Rac provides a unique molecular mechanism for the antagonism of Rho-dependent signaling through protein kinases in many cellular processes, including metastasis, immune cell chemotaxis, and neuronal development.

SUBMITTER: Gentile S

PROVIDER: S-EPMC1458818 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Rac GTPase signaling through the PP5 protein phosphatase.

Gentile Saverio S Darden Thomas T Erxleben Christian C Romeo Charles C Russo Angela A Martin Negin N Rossie Sandra S Armstrong David L DL

Proceedings of the National Academy of Sciences of the United States of America 20060320 13

We have investigated the Rac-dependent mechanism of KCNH2 channel stimulation by thyroid hormone in a rat pituitary cell line, GH(4)C(1), with the patch-clamp technique. Here we present physiological evidence for the protein serine/threonine phosphatase, PP5, as an effector of Rac GTPase signaling. We also propose and test a specific molecular mechanism for PP5 stimulation by Rac-GTP. Inhibition of PP5 with the microbial toxin, okadaic acid, blocked channel stimulation by thyroid hormone and by ...[more]

PMID: 16549782

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Project description:BackgroundProtein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.ResultsThe specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP) and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth.ConclusionsMany characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log phase growth suggests that this enzyme is involved in cell growth or its expression is controlled by metabolic or nutritional signals.

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Rac GTPase signaling through the PP5 protein phosphatase.

Publications

Rac GTPase signaling through the PP5 protein phosphatase.

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