YopH is a Yersinia protein that has protein tyrosine phosphate phosphatase activity (PTPase), is required for virulence and has homology to eukaryotic SH2 proteins and SH3 binding domains. Dr. Bliska has shown that a mutation, C403A, abolishes PTPase activity, reduces virulence, and enhances binding to tyrosine protein kinases (TPKinases). He has also shown that mutations in the SH3-binding domain block binding of the TPKinase, P53- Lyn and the adapter protein, GPRB2, but do not block binding of other phosphoproteins.YopH is delivered to the interior of the macrophage (M/0) by other Yersinia proteins and Dr. Bliska has shown that it inhibits the bacteria-induced oxidative burst. Others have shown that it inhibits phagocytosis and Dr. Bliska has shown that the PTPase activity is required for this inhibition. Dr. Bliska's hypothesis is that YopH mimics cellular PTPases and interferes with intracellular signalling in macrophages by dephosphorylating key proteins. It thus blocks the antibacterial activities of these cells. In the present application, which is a second revision of Dr. Bliska's R29 application, the investigator proposes to continue tests of this hypothesis, focussing primarily on a region of the protein that binds SH3 domains of key cellular PTKinases, chiefly P53-Lyn. The idea here is to identify the specific intracellular interprotein interaction by which YopH performs its virulence function. The basic approach is to compare the effects of mutations in this domain on binding SH3 proteins in vitro with their effect on key cellular process related to antibacterial activity such as phagocytosis and the oxidative burst. He proposes also to study certain other receptor-mediated intracellular signalling functions. The previous version received a priority in the outstanding range, but didn't quite make a fundable level, chiefly because the experiment to test whether a mutation in the SH3 binding domain eliminated (adapter- mediated) binding to phosphoproteins had not been done as a preliminary and because the only proposed test of the effects of YopH was on phagocytosis. Dr. Bliska has responded to these criticisms by performing the missing experiment and by proposing several other types of tests of YopH function in vivo. The binding experiment showed that the SH3-binding domain is required for binding one of the TPKinases, P53-Lyn, as well as the adapter protein, GRB2, but is not required for binding other phosphoproteins. Accordingly, he has found in the N-terminal 130 residues, upstream of the SH3-binding motif, homology to SH2 domains that is probably significant and will be studied as such in a proposed analysis of phosphoprotein binding. The present proposal is highly focussed and contains three specific aims: (1) to prepare and test mutants in the SH3-binding motif of YopH for their effects on the pathogenicity of Y. pseudotuberculosis (Yps) in a mouse model; (2) to analyze the intracellular activities of the mutant YopH proteins and (3)to analyze the role of the SH2-like domain near the YopH N-terminus in the binding of phosphoproteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI035175-05
Application #
2886885
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1995-07-01
Project End
2000-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Genetics
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Koblin, Beryl A; Chesney, Margaret A; Husnik, Marla J et al. (2003) High-risk behaviors among men who have sex with men in 6 US cities: baseline data from the EXPLORE Study. Am J Public Health 93:926-32
Montagna, L G; Ivanov, M I; Bliska, J B (2001) Identification of residues in the N-terminal domain of the Yersinia tyrosine phosphatase that are critical for substrate recognition. J Biol Chem 276:5005-11
Black, D S; Bliska, J B (2000) The RhoGAP activity of the Yersinia pseudotuberculosis cytotoxin YopE is required for antiphagocytic function and virulence. Mol Microbiol 37:515-27
Black, D S; Marie-Cardine, A; Schraven, B et al. (2000) The Yersinia tyrosine phosphatase YopH targets a novel adhesion-regulated signalling complex in macrophages. Cell Microbiol 2:401-14
Weidow, C L; Black, D S; Bliska, J B et al. (2000) CAS/Crk signalling mediates uptake of Yersinia into human epithelial cells. Cell Microbiol 2:549-60
Bliska, J B (2000) Yop effectors of Yersinia spp. and actin rearrangements. Trends Microbiol 8:205-8
Palmer, L E; Pancetti, A R; Greenberg, S et al. (1999) YopJ of Yersinia spp. is sufficient to cause downregulation of multiple mitogen-activated protein kinases in eukaryotic cells. Infect Immun 67:708-16
Orth, K; Palmer, L E; Bao, Z Q et al. (1999) Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersinia effector. Science 285:1920-3
Palmer, L E; Hobbie, S; Galan, J E et al. (1998) YopJ of Yersinia pseudotuberculosis is required for the inhibition of macrophage TNF-alpha production and downregulation of the MAP kinases p38 and JNK. Mol Microbiol 27:953-65
Black, D S; Montagna, L G; Zitsmann, S et al. (1998) Identification of an amino-terminal substrate-binding domain in the Yersinia tyrosine phosphatase that is required for efficient recognition of focal adhesion targets. Mol Microbiol 29:1263-74

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