With the recent clinical success of drugs targeting protein kinase activity, drug discovery efforts are focusing on the role of reversible protein phosphorylation in disease states. The activity of protein phosphatases, enzymes that oppose protein kinases, can also be manipulated to alter cellular signaling for therapeutic benefits. In this review, we present protein serine/ threonine phosphatases as viable therapeutic targets, discussing past successes, current challenges, and future strategies for modulating phosphatase activity.Numerous cellular processes, including metabolism, immune response, synaptic plasticity, cell growth and proliferation, and apoptosis, are controlled by intricate signal transduction networks composed of molecules and macromolecular protein complexes that are responsive to biological or chemical stimuli in the cell's immediate environment. A common mechanism used by cells to either propagate or terminate intracellular signal transduction pathways is reversible protein phosphorylation, whereby the addition or removal of a negatively charged phosphate can alter the conformation of a target protein and/or its interactions with other proteins. Ultimately, phosphorylation/dephosphorylation reactions affect the activity, function, half-life, or subcellular localization of the substrate; hence, the underlying molecular mechanisms controlling this reversible post-translational modification are of great physiological importance.Careful study has been afforded to the structure, function, and regulation of the enzymes that catalyze phosphorylation or dephosphorylation reactions-protein kinases and phosphatases, respectively. It is widely accepted that proper spatial and temporal regulation of both protein kinases and phosphatases is crucial for maintaining the appropriate balance of phosphorylation required for cellular homeostasis (Bauman and Scott, 2002). Because deregulation of these enzymes has been implicated in a variety of diseases (e.g., cancer, diabetes, cardiac hypertrophy, and neurodegeneration), emerging therapeutic strategies have focused on the design of drugs that affect the biological actions of kinases and phosphatases.Protein kinases have become increasingly popular drug targets, constituting ϳ30% of several pharmaceutical manufacturers' drug discovery programs (Cohen, 2002a). The approval of rapamycin (Sirolimus) for immunosuppression,

Protein serine/threonine phosphatase 2A (PP2A) activity must be tightly controlled to maintain cell homeostasis. Here, we report the identification of a previously uncharacterized mammalian protein, type 2A-interacting protein (TIP), as a novel regulatory protein of PP2A and the PP2A-like enzymes PP4 and PP6. TIP is a ubiquitously expressed protein and parallels the distribution of the PP2A catalytic subunit. Unlike its role in yeast, TIP does not interact with the mammalian homolog of type 2A-associated protein of 42 kDa (Tap42), a4, but instead associates with PP2A, PP4 and PP6 catalytic subunits independently of mammalian target of rapamycin kinase activity. Interestingly, the 20 kDa TIP splice variant TIP_i2, which lacks amino acids 173-272 of TIP's C-terminus, does not interact with PP2A; this finding indicates that residues 173-272 are important for the assembly of the TIP . phosphatase complex. In contrast to purified PP2A holoenzymes, TIP . PP2A complexes are devoid of phosphatase activity. Furthermore, alterations in the cellular levels of TIP influence the phosphorylation state of a specific protein substrate of ataxia-telangiectasia mutated (ATM)/ATM-and Rad3-related (ATR) kinases. Elevated levels of TIP result in an increase in the phosphorylation state of this protein substrate, whereas TIP-depleted cells exhibit a significant decrease in this protein's phosphorylation state, which is reversed by treatment with the PP2A inhibitor okadaic acid. These results indicate TIP is a novel inhibitory regulator of PP2A and implicate a role for TIP . PP2A complexes within the ATM/ATR signaling pathway controlling DNA replication and repair.

Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues [46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.Protein serine/threonine phosphatase 2A (PP2A) is an abundant cellular enzyme with numerous substrates that modulate a wide variety of cellular functions. Considering the multitude of cellular processes under the control of PP2A, it is not surprising that several different mechanisms exist to regulate phosphatase activity. These regulatory mechanisms include association with specific regulatory subunits and post-translational modifications of PP2Ac (i.e. phosphorylation, carboxymethylation, and ubiquitination) (1-3). Both biochemical and structural studies of PP2A have provided key mechanistic insights to explicate regulation of phosphatase holoenzyme composition and activity via phosphorylation and carboxymethylation (1,2,4,5); however, little is known about PP2Ac ubiquitination beyond the initial report demonstrating the polyubiquitination and degradation of microtubule-associated PP2Ac (3). The E3 ubiquitin ligase responsible for targeting PP2Ac for proteasome degradation

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Protein serine/threonine phosphatase (PP2A) is a major cellular enzyme implicated in the control of numerous signaling processes. The accurate measurement of PP2A activity in crude cell lysates, immune complexes, and purified preparations provides insight into the function and regulation of this essential enzyme, which, in turn, can lead to a better understanding of the signaling pathways that it modulates. The method presented here utilizes 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) and a FLEXstation for the continuous measure of PP2A activity associated with many different protein preparations. This automated fluorescence-based assay offers several distinct advantages over colorimetric and radioactive assays of phosphatase activity including (1) decreased substrate preparation time, (2) real-time kinetic data, (3) high sensitivity, and (4) the capability to analyze a wide variety of phosphatases.