The ubiquitous transcription factor Yin Yang 1 (YY1) is known to have a fundamental role in normal biologic processes such as embryogenesis, differentiation, replication, and cellular proliferation. YY1 exerts its effects on genes involved in these processes via its ability to initiate, activate, or repress transcription depending upon the context in which it binds. Mechanisms of action include direct activation or repression, indirect activation or repression via cofactor recruitment, or activation or repression by disruption of binding sites or conformational DNA changes. YY1 activity is regulated by transcription factors and cytoplasmic proteins that have been shown to abrogate or completely inhibit YY1-mediated activation or repression; however, these mechanisms have not yet been fully elucidated. Since expression and function of YY1 are known to be intimately associated with progression through phases of the cell cycle, the physiologic significance of YY1 activity has recently been applied to models of tumor biology. The majority of the data are consistent with the hypothesis that YY1 overexpression and/or activation is associated with unchecked cellular proliferation, resistance to apoptotic stimuli, tumorigenesis and metastatic potential. Studies involving hematopoetic tumors, epithelial-based tumors, endocrine organ malignancies, hepatocellular carcinoma, and retinoblastoma support this hypothesis. Molecular mechanisms that have been investigated include YY1-mediated downregulation of p53 activity, interference with poly-ADP-ribose polymerase, alteration in c-myc and nuclear factor-kappa B (NF-kappaB) expression, regulation of death genes and gene products, and differential YY1 binding in the presence of inflammatory mediators. Further, recent findings implicate YY1 in the regulation of tumor cell resistance to chemotherapeutics and immune-mediated apoptotic stimuli. Taken together, these findings provide strong support of the hypothesis that YY1, in addition to its regulatory roles in normal biologic processes, may possess the potential to act as an initiator of tumorigenesis and may thus serve as both a diagnostic and prognostic tumor marker; furthermore, it may provide an effective target for antitumor chemotherapy and/or immunotherapy.

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Protein-tyrosine phosphatases can exhibit stringent substrate specificity in vivo, although the molecular basis for this is not well understood. The three-dimensional structure of the catalytically inactive protein-tyrosine phosphate 1B (PTP1B)/C215S complexed with an optimal substrate, DADEpYL-NH 2 , reveals specific interactions between amino acid residues in the substrate and PTP1B. The goal of this work is to rigorously evaluate the functional significance of Tyr 46 , Arg 47 , Asp 48 , Phe 182 , and Gln 262 in substrate binding and catalysis, using site-directed mutagenesis. Combined with structural information, kinetic analysis of the wild type and mutant PTP1B using p-nitrophenyl phosphate and phosphotyrosine-containing peptides has yielded further insight into PTP1B residues, which recognize general features, as well as specific properties, in peptide substrates. In addition, the kinetic results suggest roles of these residues in E-P hydrolysis, which are not obvious from the structure of PTP1B/peptide complex. Thus, Tyr 46 and Asp 48 recognize common features of peptide substrates and are important for peptide substrate binding and/or E-P formation. Arg 47 acts as a determinant of substrate specificity and is responsible for the modest preference of PTP1B for acidic residues NH 2 -terminal to phosphotyrosine. Phe 182 and the invariant Gln 262 are not only important for substrate binding and/or E-P formation but also important for the E-P hydrolysis step.Protein-tyrosine phosphorylation is a universal mechanism employed for the regulation of cellular processes such as proliferation, differentiation, motility, cell-cell interactions, metabolism, gene transcription, and the immune response (1, 2). The propagation and termination of signaling events controlling these cellular processes are determined by the level of phosphorylated proteins in a cell. The phosphorylation level, in turn, is maintained in an exquisite balance by the reciprocal activities of protein-tyrosine kinases and phosphatases. Thus, in addition to the study of protein-tyrosine kinases, one can appreciate the need to further characterize the dephosphorylation reaction catalyzed by the protein-tyrosine phosphatases (PTPases). 1Much is known about the catalytic mechanism of the PTPases (3). However, the molecular basis for PTPase substrate specificity is not well understood and remains a major unresolved issue in the field. The PTPase family is presently composed of approximately 100 enzymes, which can be either transmembrane (receptor-like) or intracellular (cytoplasmic). Membership in this family of enzymes requires the presence of the PTPase signature motif, (H/V)CX5R(S/T), housed within the catalytic domain. Outside this shared catalytic domain are various targeting and localization domains, which may be utilized for controlling and restricting PTPase substrate specificity. There have been relatively few biochemical analyses of the mechanisms that govern PTPase substrate specificity, although recent genetic and biochemical evidence sugg...

The hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases (PTPases) was studied with the aim of providing a mechanistic framework for the reactions of this important class of substrate analogues. O-Arylphosphorothioates are hydrolyzed 2 to 3 orders of magnitude slower than O-aryl phosphates by PTPases. This is in contrast to the solution reaction where phosphorothioates display 10-60-fold higher reactivity than the corresponding oxygen analogues. Kinetic analyses suggest that PTPases utilize the same active site and similar kinetic and chemical mechanisms for the hydrolysis of O-arylphosphorothioates and O-aryl phosphates. Thio substitution has no effect on the affinity of substrate or product for the PTPases. Brønsted analyses suggest that like the PTPase-catalyzed phosphoryl transfer reaction the transition state for the PTPase-catalyzed thiophosphoryl transfer is highly dissociative, similar to that of the corresponding solution reaction. The side chain of the active-site Arg residue forms a bidentate hydrogen bond with two of the terminal phosphate oxygens in the ground state and two of the equatorial oxygens in a transition state analog complex with vanadate Proc. Natl. Acad. Sci. USA 93, 2493USA 93, -2498 Zhang, M. et al. (1997) Biochemistry 36, 15-23; Pannifer et al. (1998) J. Biol. Chem. 273, 10454-10462]. Replacement of the active-site Arg409 in the Yersinia PTPase by a Lys reduces the thio effect by 54-fold, consistent with direct interaction and demonstrating strong energetic coupling between Arg409 and the phosphoryl oxygens in the transition state. These results suggest that the large thio effect observed in the PTPase reaction is the result of inability to achieve precise transition state complementarity in the enzyme active site with the larger sulfur substitution. Protein tyrosine phosphatases (PTPases)1 catalyze the removal of the phosphoryl group from aryl phosphates and phosphotyrosine in peptides/proteins. The PTPase superfamily includes the classical tyrosine-specific PTPases, the dual specificity phosphatases, and the low molecular weight phosphatases. These three groups of phosphatases share the signature motif (H/V)C(X) 5 R(S/T) and other key structural features that are important for catalysis and are believed to utilize a common mechanism to effect catalysis (1). In this mechanism (Figure 1), the side chain of the active-site Cys residue serves as a nucleophile to accept the phosphoryl group from the substrate and form a kinetically competent cysteinyl phosphate intermediate (2, 3). The active-site Arg residue interacts with the phosphoryl moiety of the substrate and plays a role in both substrate binding and transition state stabilization (4). To facilitate substrate turnover, PTPases also employ an invariant Asp residue, which acts as a general acid by protonating the ester oxygen of the leaving group leading to the formation of the cysteinyl phosphate intermediate (5). The phosphoenzyme intermediate is subsequently hydrolyzed by a water molecule, which i...