Control of cell signaling by PTPases

1. Control of cell signaling by PTPases 

2. 1) Typical protein: MW 50-100,000 Phosphate: MW 80, yet drastic effects: e.g. there are other proteins that have domains that specifically bind to Y-P in specific environments 2) estimated 30% of proteins modif. in this way 3) Kinases: discovered from 1980s on (roughly) Phosphatases: research wave started 10 years later1) Typical protein: MW 50-100,000 Phosphate: MW 80, yet drastic effects: e.g. there are other proteins that have domains that specifically bind to Y-P in specific environments 2) estimated 30% of proteins modif. in this way 3) Kinases: discovered from 1980s on (roughly) Phosphatases: research wave started 10 years later

3. 1) Typical protein: MW 50-100,000 Phosphate: MW 80, yet drastic effects: e.g. there are other proteins that have domains that specifically bind to Y-P in specific environments 2) estimated 30% of proteins modif. in this way 3) Kinases: discovered from 1980s on (roughly) Phosphatases: research wave started 10 years later1) Typical protein: MW 50-100,000 Phosphate: MW 80, yet drastic effects: e.g. there are other proteins that have domains that specifically bind to Y-P in specific environments 2) estimated 30% of proteins modif. in this way 3) Kinases: discovered from 1980s on (roughly) Phosphatases: research wave started 10 years later

6. Why study Protein Tyrosine Phosphatases (PTPs) ?

9. R254 is one of the residues that is unique to PTP1b (also T-cell PTP) and that helps shape the 2nd PTyr binding pocket. Pocket can be exploited using bi-dentate inhibitors R24 and G259 are unique to PTP1B G259: small residue is responsible for broad substrate-specificity of PTP1B (as opposed to e.g. LAR, PTPa, where there is a bulky residue)(make this G in RPTPa makes RPTPa more promiscuous)(gateway residue) R47 is plastic, and also helps to accommodate diverse substrates The SHP specificity region is involved in peptide substrate binding in SHP1 (and is different in SHP2). Domain-swapping expts also implicate this region in the substrate spec. of SHP1 vs. SHP2 D48 in PTP1B is an asparagine in most other PTPs > basic Nitrogen here in inhibitor allowed spec. for PTP1BR254 is one of the residues that is unique to PTP1b (also T-cell PTP) and that helps shape the 2nd PTyr binding pocket. Pocket can be exploited using bi-dentate inhibitors R24 and G259 are unique to PTP1B G259: small residue is responsible for broad substrate-specificity of PTP1B (as opposed to e.g. LAR, PTPa, where there is a bulky residue)(make this G in RPTPa makes RPTPa more promiscuous)(gateway residue) R47 is plastic, and also helps to accommodate diverse substrates The SHP specificity region is involved in peptide substrate binding in SHP1 (and is different in SHP2). Domain-swapping expts also implicate this region in the substrate spec. of SHP1 vs. SHP2 D48 in PTP1B is an asparagine in most other PTPs > basic Nitrogen here in inhibitor allowed spec. for PTP1B

17. -test a battery of PTP KOs: see if YP of protein X is increased -IP protein X, see if a PTP is co-precipitated, by doing in vitro deP assay -do mass-spec on assoc. proteins -before IP, inactivated the PTPs by H2O2, perhaps they will bind the substrate ?-test a battery of PTP KOs: see if YP of protein X is increased -IP protein X, see if a PTP is co-precipitated, by doing in vitro deP assay -do mass-spec on assoc. proteins -before IP, inactivated the PTPs by H2O2, perhaps they will bind the substrate ?

19. The size and complexity of the Protein Tyrosine Phosphatase (PTP) familyequal that of the tyrosine kinases

22. Cbl > degradation ((role of Src in YP-ing and activating Cbl ?) SHIPCbl > degradation ((role of Src in YP-ing and activating Cbl ?) SHIP

43. heterodimerization !heterodimerization !

44. -Confirm that dim. occurs: -biochem cross-linking -FRET -induce dimerization artificially and look for effects -EGFR/CD45 chimera -antibodies -FKBP1012 etc. -mutagenesis: mutate wedge Jiang et al A. Weiss in EGFR/CD45 chimera -in vivo: knock-in mut into wedge-Confirm that dim. occurs: -biochem cross-linking -FRET -induce dimerization artificially and look for effects -EGFR/CD45 chimera -antibodies -FKBP1012 etc. -mutagenesis: mutate wedge Jiang et al A. Weiss in EGFR/CD45 chimera -in vivo: knock-in mut into wedge

46. Figure 1. Ribbon diagram of the receptor PTP� D1 dimer. The molecular dyad axis is indicated by an arrow. The catalytic site cysteine (Cys1095), the helix-turn-helix segment (1, 2), and the N-terminal -strand (x) that forms a -sheet with y within RPTPs D1 are indicated within one subunit (left). The 8 and 10 strands participate in the dimer interface of RPTP� D1. C atoms of the equivalent catalytic site residues that form the dimer interface of RPTP D1 are shown as spheres and labeled. Figure was drawn using MOLSCRIPT (22). Figure 1. Ribbon diagram of the receptor PTP� D1 dimer. The molecular dyad axis is indicated by an arrow. The catalytic site cysteine (Cys1095), the helix-turn-helix segment (1, 2), and the N-terminal -strand (x) that forms a -sheet with y within RPTPs D1 are indicated within one subunit (left). The 8 and 10 strands participate in the dimer interface of RPTP� D1. C atoms of the equivalent catalytic site residues that form the dimer interface of RPTP D1 are shown as spheres and labeled. Figure was drawn using MOLSCRIPT (22).

56. binding may be cis binding may reflect PTP acting as ligand rather than as receptorbinding may be cis binding may reflect PTP acting as ligand rather than as receptor

57. Ligands can affect -phosphorylation (YP, auto-de-P ?) -dimerization (pos. or neg.) -sensitivity to redox-regulation -heterodimerization -association with other proteins , or substrates (liprins, �) Ligands can affect -phosphorylation (YP, auto-de-P ?) -dimerization (pos. or neg.) -sensitivity to redox-regulation -heterodimerization -association with other proteins , or substrates (liprins, �)