Zhongsen Zhang

Johns Hopkins University, Baltimore, MD, United States

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Publications (5)42.7 Total impact

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    ABSTRACT: The low molecular weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitously expressed enzyme with several proposed roles in cell signaling. Previously, two tyrosine phosphorylation modifications of LMW-PTP at sites Tyr-131 and Tyr-132 in response to growth factor stimulation have been mapped and suggested to stimulate LMW-PTP phosphatase activity. Biochemical analysis of tyrosine phosphorylation of a tyrosine phosphatase is challenging because of the intrinsic instability of these modifications. Here we used expressed protein ligation to site-specifically incorporate a phosphotyrosine mimic (phosphonomethylenephenylalanine, Pmp) at the Tyr-131 and Tyr-132 positions and measured the catalytic activity of these semisynthetic LMW-PTPs. The phosphonate-modified LMW-PTPs were 10- to 23-fold less active in dephosphorylating phosphotyrosine peptides derived from the PDGF receptor and p190RhoGap, two putative cellular substrates. These findings suggest the first example of a tyrosine phosphatase that is inhibited by tyrosine phosphorylation and provide a new model for the regulation of LMW-PTP and its role in cell adhesion.
    Journal of the American Chemical Society 05/2006; 128(13):4192-3. · 11.44 Impact Factor
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    ABSTRACT: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) controls daily changes in the production and circulating levels of melatonin. Here, the significance of the phosphorylation of AANAT was studied using a semisynthetic enzyme in which a nonhydrolyzable phosphoserine/threonine mimetic, phosphonomethylenealanine (Pma), was incorporated at position 31 (AANAT-Pma31). The results of studies in which AANAT-Pma31 and related analogs were injected into cells provide the first direct evidence that Thr31 phosphorylation controls AANAT stability in the context of the intact cells by binding to 14-3-3 protein. These findings establish Thr31 phosphorylation as an essential element in the intracellular regulation of melatonin production. The application of Pma in protein semisynthesis is likely to be broadly useful in the analysis of protein serine/threonine phosphorylation.
    Nature Structural Biology 01/2004; 10(12):1054-7.
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    ABSTRACT: Protein phosphorylation catalyzed by protein kinases plays a critical role in cellular signaling. Here we review several chemical approaches to understanding protein kinases and the consequences of protein phosphorylation. We discuss the design of bisubstrate analogue inhibitors based on a dissociative transition state, the development of reagents for cross-linking protein kinases with their substrates, the chemical rescue of mutant protein tyrosine kinases, and the application of expressed protein ligation to understanding protein phosphorylation.
    Accounts of Chemical Research 07/2003; 36(6):444-52. · 24.35 Impact Factor
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    Kara A Scheibner, Zhongsen Zhang, Philip A Cole
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    ABSTRACT: Determination of protein oligomerization state can be technically challenging. We have combined the methods of expressed protein ligation (EPL) and fluorescence resonance energy transfer (FRET) for the analysis of protein homo-oligomerization states. We have attached fluorescein (donor) and rhodamine (acceptor) chromophores via dipeptide linkages to the C-termini of three recombinant proteins and examined the potential for FRET between mixtures of these semisynthetic proteins. The known protein dimer (glutathione S-transferase) showed evidence of FRET and the known protein monomer (SH2 domain phosphatase-1) did not display FRET. Using this method, the previously uncharacterized circadian rhythm enzyme, serotonin N-acetyltransferase, displayed significant FRET, indicating its likely propensity for dimerization or more complex oligomerization. These results establish the potential of the union of EPL and FRET in the analysis of protein-protein interactions and provide insight into the unusual enzymatic behavior of a key circadian rhythm enzyme.
    Analytical Biochemistry 07/2003; 317(2):226-32. · 2.31 Impact Factor
  • Zhongsen Zhang, Kui Shen, Wei Lu, Philip A Cole
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    ABSTRACT: The protein-tyrosine phosphatase SHP-1 plays a variety of roles in the "negative" regulation of cell signaling. The molecular basis for the regulation of SHP-1 is incompletely understood. Whereas SHP-1 has previously been shown to be phosphorylated on two tail tyrosine residues (Tyr(536) and Tyr(564)) by several protein-tyrosine kinases, the effects of these phosphorylation events have been difficult to address because of the intrinsic instability of the linkages within a protein-tyrosine phosphatase. Using expressed protein ligation, we have generated semisynthetic SHP-1 proteins containing phosphotyrosine mimetics at the Tyr(536) and Tyr(564) sites. Two phosphonate analogues were installed, phosphonomethylenephenylalanine (Pmp) and difluorophosphonomethylenephenylalanine (F(2)Pmp). Incorporation of Pmp at the 536 site led to 4-fold stimulation of the SHP-1 tyrosine phosphatase activity whereas incorporation at the 564 site led to no effect. Incorporation of F(2)Pmp at the 536 site led to 8-fold stimulation of the SHP-1 tyrosine phosphatase activity and 1.6-fold at the 564 site. A combination of size exclusion chromatography, phosphotyrosine peptide stimulation studies, and site-directed mutagenesis led to the structural model in which tyrosine phosphorylation at the 536 site engages the N-Src homology 2 domain in an intramolecular fashion relieving basal inhibition. In contrast, tyrosine phosphorylation at the 564 site has the potential to engage the C-Src homology 2 domain intramolecularly, which can modestly and indirectly influence catalytic activity. The finding that phosphonate modification at each of the 536 and 564 sites can promote interaction with the Grb2 adaptor protein indicates that the intramolecular interactions fostered by post-translational modifications of tyrosine are not energetically strong and susceptible to intermolecular competition.
    Journal of Biological Chemistry 03/2003; 278(7):4668-74. · 4.60 Impact Factor