Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

Abstract

Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-␤ and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93 Å resolution. The polypeptide chain adopts the typical ␣␤␣ PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.Protein phosphatases and kinases together help control a host of cellular functions (1). Reversible phosphorylation of tyrosine, serine, and threonine side chains represents a fundamental mode of cellular signal transduction, regulating cell growth and differentiation. Protein phosphatases, unlike the protein kinase superfamily, can be classified as members of separate superfamilies that are mechanistically and structurally distinct from one another (2). Based on the substrate specificity toward phospho-seryl/threonyl (pSer/pThr) 2 or tyrosyl residues (pTyr), they are divided into two families: protein serine/threonine phosphatases and protein-tyrosine phosphatases (PTPs). The so-called dual specificity phosphatases (DSPs) dephosphorylate both pTyr and pSer/pThr and belong to class I cysteine-based PTPs. Cys-based PTPs and DSPs share the conserved signature motif HCXXGXXRS(T) and exhibit similar mechanisms of catalysis, involving a two-step process that proceeds via formation of a cysteinyl-phosphate enzyme-substrate intermediate (3).Mitogen-activated protein kinase (MAPK) activation requires phosphorylation at threonine and tyrosine with a characteristic TY/TXY motif found within the enzyme activation loop. This reversible process is mediated by either MAPK phosphatases (MKPs) or dual specificity MKPs (DSPs) (4). DSPs control MAPK signaling via dephosphorylation and exhibit feedback regulation (5). There are ϳ65 human genes encoding various DSPs with low sequence similarity with respect to one another (2). The gene encoding dual specificity protein phosphatase 23 (DUSP23) is located on human chromosome 1q23 (6). DUSP23 (originally erroneously termed DUSP25) encodes a 150-residue protein tha...

Structural basis for the glucan phosphatase activity of Starch Excess4

“…Many dual specificity phosphatases contain little more than a DSP domain and can be readily expressed in Escherichia coli (38)(39)(40)(41). The DSP of SEX4 cannot be independently expressed, and we hypothesized that the intimate association of the C-terminal domain with the DSP underlies this phenomenon.…”

Dimeric Quaternary Structure of the Prototypical Dual Specificity Phosphatase VH1

“…VH1-like DSPs share a common catalytic mechanism, which is mediated by a catalytic triad consisting of a cysteine, an arginine, and an aspartic acid, usually present in the context of an extended consensus motif (4). The structural organization of the minimum catalytic core of VH1-like DSPs is known from the crystal structures of several members of the VH1-like family, such as VHZ (5) and VHR (6). All known DSPs share a common topology with members of the classical protein-tyrosine phosphatases, with the most marked structural difference being in the architecture of the active site.…”

“…The Dimerization Interface-The dimeric quaternary structure of VH1 is kept in place by an extended domain swap of the N-terminal helix ␣1 (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. In a dimer of VH1, the two N-terminal swapped helices ␣1A and ␣1B cross each other at an angle of ϳ95°.…”

Dimerization of Vaccinia Virus VH1 Is Essential for Dephosphorylation of STAT1 at Tyrosine 701

“…In both cases, the structure revealed a conserved PTP-like fold similar to that previously reported for VHZ (21) and VHR (22). The VH1 active site consists of a shallow catalytic cleft only ϳ6 Å deep, which can accommodate both phosphotyrosine and phosphothreonine/serine residues.…”