Protein Phosphatase Inhibitors
Phosphorylation and dephosphorylation of structural and regulatory proteins are major intracellular control mechanisms in eukaryotes. Protein kinases transfer a phosphate from ATP to a specific protein, typically at serine, threonine, or tyrosine residues. Phosphatases remove the phosphoryl group and restore the protein to its original dephosphorylated state. Hence, the phosphorylation-dephosphorylation cycle can be regarded as a molecular "on-off" switch.
Protein phosphatases (PPs) have been classified into three distinct categories: serine/threonine (Ser/Thr)-specific, tyrosine-specific, and dual-specificity phosphatases. Based on biochemical parameters, substrate specificity, and sensitivity to various inhibitors, Ser/Thr protein phosphatases are divided into two major classes. Type I phosphatases, which include PP1, can be inhibited by two heat-stable proteins known as Inhibitor-1 (I-1) and Inhibitor-2 (I-2). They preferentially dephosphorylate the β-subunit of phosphorylase kinase. Type II phosphatases are subdivided into spontaneously active (PP2A), Ca2+ -dependent (PP2B), and Mg2+-dependent (PP2C) classes of phosphatases. They are insensitive to heat-stable inhibitors and preferentially dephosphorylate the α-subunit of phosphorylase kinase.
Protein tyrosine phosphatases (PTPs) are relatively recent additions to the phosphatase family. They remove phosphate groups from phosphorylated tyrosine residues of proteins. PTPs display diverse structural features and play important roles in the regulation of cell proliferation, differentiation, cell adhesion and motility, and cytoskeletal function. They are either transmembrane receptor-like PTPs or cytosolic enzymes. Each PTP contains a highly conserved catalytic domain of about 240 residues that contains highly conserved arginine and cysteine residues at the catalytic domain. The diversity of PTPs is primarily due to the variety of non-catalytic regulatory sequences and targeting domains attached to both N- and C-termini.
Another category of protein phosphatases is the dual specificity phosphatases (DSPs), which play a key role in the dephosphorylation of MAP kinases. Hence, they are also termed as MAP kinase phosphatases (MKPs). On the basis of predicted structures, MKPs have been divided into three subgroups. Group I contains DSP1, DSP2, DSP4, and DSP5; group II enzymes are DSP6, DSP7, DSP9, and DSP10; and group III consists of DSP8 and DSP16. All the DSPs share strong amino-acid sequence homology in their catalytic domains. The catalytic domain contains a highly conserved consensus sequence DX26(V/L)X(V/ I)HCXAG(I/V) SRSXT(I/V) XXAY(L/I)M, where X could be any amino acid. The three underlined amino acids are reported to be essential for the catalytic activity of DSPs. The cysteine is required for the nucleophilic attack on the phosphorus of the substrate and the formation of the thiol-phosphate intermediate. The conserved arginine binds the phosphate group of phosphotyrosine or phosphothreonine, enabling transition-state stabilization, and the aspartate enhances catalysis by protonating oxygen on the departing phosphate group. All DSPs contain two conserved regions, known as the CH2 domains, at their amino terminus, which are involved in substrate binding. In addition, they contain a MAP kinase-docking site at the amino terminus that consists of a cluster of positively charged amino acids. The corresponding docking site on MAP kinases consists of negatively charged residues indicating that electrostatic interactions are involved in binding of MAP kinases and MKPs. The group III DSPs also have an extended carboxy terminus containing PEST sequences (abundant in proline, glutamate, serine and threonine) that are commonly found in rapidly degrading proteins. Removal of PEST sequences results in their stabilization.
DSP1, DSP2, DSP4, and DSP5 are located exclusively in the nucleus, whereas DSP6, DSP7, and DSP16 are predominantly expressed in the cytoplasm. DSP8, DSP9, and DSP10 exhibit both cytoplasmic and nuclear expression. Newly reported DSP18 and DSP21 are localized in the mitochondria.
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