Biochem 503 December 1, 2008 Protein Tyr Phosphatases assigned reading: Stoker (2005) J. Endocrin. 185:19-33 Tonks (2006) Nature Reviews MCB 7: 833-846
Dec 24, 2015
Biochem 503 December 1, 2008
Protein Tyr Phosphatases
assigned reading:
Stoker (2005) J. Endocrin. 185:19-33
Tonks (2006) Nature Reviews MCB 7: 833-846
Phosphotyrosyl Protein Phosphatase PTPP (original name)
Brautigan DL, Bornstein P, Gallis B. Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn.J Biol Chem. 1981 Jul 10;256(13):6519-22. PMID: 6165721
Swarup G, Cohen S, Garbers DL. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate.Biochem Biophys Res Commun. 1982 Aug;107(3):1104-9.
Swarup G, Speeg KV Jr, Cohen S, Garbers DL. Phosphotyrosyl-protein phosphatase of TCRC-2 cells.J Biol Chem. 1982 Jul 10;257(13):7298-301.
History of P-Tyr Phosphatases1981-1982 First description of P-Tyr specific phosphatase
activity in A431 cell membrane fractions. EGF-R substrateSelective inhibition by Zn2+…… or vanadate
1983-1986 Development of assays using P-Tyr proteins: histone, poly[Glu:Tyr], denatured BSA and lysozyme.
Biochemical fractionations of membrane and cytosolic PTPs of 35 kDafrom various tissues and cell lines.
Reaction with EGFR, InsR, IGF-R, requirement for SH.
1987-1988 purification of PTPs from human placenta (Tonks and Fischer)
1988-1989 partial peptide sequence of PTP1B - Unique phosphatase (Charbonneau, Tonks, Walsh & Fischer)
1990 demonstration of CD45 as a PTPase
Unexpected sequence similarity to existing protein CD-45CD45 already known as abundant lymphocyte surface antigen
Nick TonksCSHL
PTP1B
CD45, aka LCA, B220
40% 33%
1990’s cloning of multiple PTPs
Jack Dixon Purdue; U of Michigan; now at UCSD and CSO of HHMI(National Academy of Sciences USA)
showed Phospho-Cys intermediate in PTPsactive site sequence motif used to find distant relativesVHR viral PTP related to VH1 DSPYOP51 Yersina virulence genePTEN is a lipid phosphatase for PIP3
Ben Neel Harvard Med. School, now at Ontario Cancer Inst, Toronto
ER localization of PTP1Bcloning and properties of SH2-PTPsmotheaten mouse (SHP hypomorph)PTP1B knockout mouse
1990’s 3D X-ray structures of PTPs - VH1, PTP1BKnockout mouse for PTP1B
PTP1B knockout mousecritical test of PTP as drug target for diabetes
David BarfordOxford England now ICR
Michel TremblayMcGill Univ., Montreal
Mark SaperUniv. of Michigan
Vanadate complex with VHR
mimics phospho-enzyme
PTP1B structure and discovery of
Cys Oxidation to cyclic sulfenamide
PTP-like Phosphatases in The Human Genome
Cell (2004) 117:699
transmembrane
cytoplasmic
assorted others
Crystal structures of six PTP domains show a conserved fold and C-backbone
N-terminal
Central -helix
Superimposition of PTP1B (magenta), RPTP (gray), RPTP (red), LAR (blue), SHP1 (green) and SHP2 (yellow).
Andersen et al Mol. Cell. Biol. 2001
Phe180Phe180
Asp181Asp181
Tyr46Tyr46
Gln262Gln262
Arg221Arg221
WPD-loop
pTyr-loop
Q-loop
PTP-loop
Cys215Cys215
Phosphotyrosine (Substrate)
The PTP1B active site
Protein Tyr Phosphatases (PTPs)
A. Common Enzyme Mechanism and Kinetics
1. specificity for P-Tyr, vs, P-Ser or P-Thra large (long, aromatic) sidechain P-Tyr is a high energy phosphoester, used as intermediate in
topoisomerasesunusual KM < 1 uM, implies special interactions - trapping mechanism
2. signature catalytic site sequence HCxxGxxR with essential Cys SH group, and R residue
4. phospho-enzyme intermediate Cys-S-PO4
a. vanadate mimics the transition state of phosphate b. proton transfer to leaving group TyrOH by D181 is essential c. hydrolysis and product (PO4) release is rate-limiting, d. 1000 sec-1 reduced to 0.02 sec-1 by D181A mutation e. either Cys to Ser or Asp to Ala mutations make inactive PTP
5. reversible oxidation of Cys to regulate activity forms cyclic cysteinyl-sulfenyl-amide, can be reduced by thiols
OH S OH CH2 CH2
HN-CH-C-NH-CH-C-NH O O
ROS or H2O2
Cys-SH Cys-SOH
GSH
B. Sub-Families of Tyr Phosphatases (PTPs)
1. Transmembrane PTPs - the prototype CD-45
a. common features (most)1. single TM helix to span membrane2. double PTP domain, with activity in N terminal (D1) domain3. large extracellular domains, related to cell-cell adhesion4. inhibited by dimerization? Oxidation? 5. activators of src kinases by Tyr527 dephosphorylation
b. differences
1. tissue and developmental expression2. substrate specificity, but few targets known
knockouts and trapping mutants3. inhibitors of active sites as pharmaceuticals
Cys-SH
Cys-SH
Cys-SH
Cys-SNHROS
active inactive
Regulation of Transmembrane PTPs by Oxidation of D2 Domains
2. Cytosolic PTPs, the prototype PTP1B
a. common features 1. single PTP domain, plus targeting sequences2. specificity for P-Tyr vs. P-Ser3. Phospho-Cys-enzyme intermediate4. Substrate trap by conformational movement5. Oxidation-reduction control mechanism
b. differences1. tissue expression2. specificity for substrates3. Inhibition by small molecules
Liver
Muscle
Muscle
Tyrosine Phosphorylation of Insulin ReceptorResistance to High Fat Diet
-/-,+/-
-/-,+/-
+/+
+/+
SHP2 phosphatase regulated by P-Tyr binding:either intrasteric or intermolecular
PTP
SH2
SH2PTP
SH2 SH2
P-Tyr P-Tyr
Y542
Y580
active
SHP2 phosphataseActivating mutations in Noonan’s Syndrome
PTP
SH2
SH2
Split open to expose interface
3. Dual Specificity Phosphatases, the prototype VH1
a. mechanism common with PTPs, i.e. Cys-Phosphate but shallow active site to accommodate P-Ser/P-Thr
b. the MKPs (DUSPs), MAP kinase phosphatasesbinds to MAPK at site in N terminal domainand this activates the MKP C terminal catalytic domain
several members : CL100, MKP1, 2, 3, 4,
c. the cdc25 family of CDK phosphataseslow activity phosphatase with extreme specificitylarge inhibitory domain, activated by phosphorylationnot really a family member- it’s like Rhodanase(??)
Dual-Specificity Phosphatase: DUSPs MAPK PPase Catalytic domain reacts with pTyr-X-pThr
Cys…Arg….Asp C R D
4. Other Cys-dependent Phosphatases
a. enzymes that share the catalytic site motif HCxxxRdiscovered by sequence searches
b. PTEN mutated in many human tumors.lipid phosphatase, removes P from PIP3 to reverse action of PI3Kresults in elevated PIP3 and activation of Akt/PKB - survival signal
c. Myotubularin a family of proteins, other PIP3 phosphatases
d. cdc14 phosphatase dephosphorylates Thr in CDK activation loop
e. Slingshot dephosphorylates cofilin at Ser3 to regulate actin