Protein Phosphatases

1. Protein Phosphatases Protein Motifs Session 7th December 2009 Kirsten McKay

2. Protein Phosphatase Protein phosphatases • Enzymes that catalyse the removal of phosphate groups from residues • Primarily Serine (86%), Threonine (12%) and Tyrosine (2%) – hydroxyl groups • Have opposite action from Kinases and Phosphorylases • Intricate regulation of many cellular signalling paths

3. Ser/Thr Protein Phosphatases • Removal of phosphate groups from serine or threonine residues (PPPs and PPMs) • Phosphoprotein phosphatases (PPPs) • PP1, PP2A, PP2B (PP3 or calcineurin), PP4, PP5; heterodimeric • Phosphoprotein phosphatases metal-dependent (PPMs) • PP2C, mitochondrial phosphatases • Monomeric, dependent upon metal ions Mg2+ or Mn2+ • Convergent evolution

4. Calcineurin • Heterodimer of a catalytic subunit (calcineurin A) and a regulatory Ca2+ binding subunit (calcineurin B) • Calcium-dependent protein phosphatase • Important in Graft versus Host Disease

5. Protein Tyrosine Phosphatases • PTP superfamily, subclassified • Class I • Classical receptor – Tyr-specific • Non-receptor/soluble – Tyr-specific • Dual-specificity – Ser/Thr as well as Tyr activity • Class II • Low molecular weight PTP • Class III • Cell cycle regulators CDC25A, B, C (also DSPs) • Class IV • Aspartate-based PTPs, eg Haloacid dehalogenase

6. Class I PTPs • Unique signature motif HC(X)5R • Cysteine residue acts as nuceophile to catalyse reaction • Arginine residue binds oxygen atoms of phosphate group • These enzymes are very important in the control of cell growth, proliferation, differentiation and transformation. • PTPN11 is a clinically important example of a non-receptor PTP

7. PTPN11 • PTPN11 gene on ch12 encodes SHP-2 protein • Contains two Src homology 2 (SH2) domains and a PTP domain • Interactions between SH2 and PTP domains maintains protein in inactive state • Binding by SH2 domain leads to activation of the PTP domain • Activates the MAPK pathway by dephosphorylation of unknown substrate, GRB2?

8. Noonan syndrome (NS) • Clinical diagnosis based upon short stature, dysmorphic facies and congenital heart defects • Widely variable phenotype • Autosomal dominant or sporadic • ~50% of NS patients have missense mutations in PTPN11 • Other patients have mutations in KRAS (causing hyperactive protein), RAF1 and SOS1

9. Noonan syndrome continued • Genotype – Phenotype correlation • PTPN11 mutations associated with pulmonary stenosis • Those without PTPN11 mutations are associated with cardiomyopathy • ¼ of mutations are c.922A>G p.Asn308Asp – often familial • c.218C>T p.Thr73Ile is associated with a predisposition to myeloproliferative disorders

10. LEOPARD syndrome (LS) • Lentigines (patches of hyperpigmented skin) • ECG conduction abnormalities • Ocular hypertelorism • Pulmonary stenosis • Abnormal genitalia • Retarded growth • Deafness • Much rarer • AD or sporadic with variable penetrance • 90% of patients have mutation in PTPN11 gene

11. LEOPARD syndrome continued • 11 specific missenses in PTPN11 have been described assoc with LS • Some genotype/phenotype correlations in LS • Small number of patients with mutations in RAF1 gene (Tyrosine kinase) • NS and LS overlap clinically with Cardio-Facio-Cutaneous (CFC) syndrome and Costello syndrome (CS) • Short stature, cardiac defects, facial dysmorphisms

12. Disorders of Ras-MAPK pathway • Costello – mutations in HRAS • CFC syndrome – mutations in KRAS, BRAF, MEK1, MEK2 • Noonan – mutations in PTPN11, SOS and KRAS • Leopard – mutations in PTPN11 and RAF1

13. PTPN11– several disorders • In NS, mutations are clustered in region important for interaction between SH2 and PTP domains and result in constitutively active SHP-2 protein • In LS, mutations in PTP domain and result in reduced SHP-2 protein activity, however perhaps not complete loss of function as mutations are missense rather than truncating. ?dominant negative or ?gain of function • Somatic activating mutations have been identified in cases of juvenile myelomonocytic leukaemia (JMML), MDS, AML, B-ALL, solid tumours etc

14. Class III PTPs – CDC25 family • These are dual-specificity phosphatases (DSPs) – can target Ser/Thr or Tyr res. • Important regulators of the cell cycle by dephosphorylation (activation) of CyclinB/Cdk1 complex • Progression through G2/M phase in absence of DNA damage signals • Intricate feedback loops involve Wee1/Myt1 kinases and CDC25 phosphatases • Targets for anti-cancer therapy

15. PTEN • 'phosphatase and tensin homolog deleted on chromosome ten’ • Another dual-specificity phosphatase (DSP) • Tumour suppressor gene that down-regulates the PI3K/Akt pathway and causes cell cycle arrest

16. PTEN continued • Inherited mutations result PTEN hamartoma tumor syndrome (PHTS) • Cowden disease, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, Proteus-like syndrome • Cowden disease is assoc with risk of malignancy • LOF – missense, nonsense, splice-site, indels, promoter, large deletions • Geemline mutations in PTEN are also associated with macrocephaly, autistic-spectrum disorder and developmental delay/mental retardation. • Somatic PTEN mutations are frequently found in endometrial, breast and prostate cancer

17. Alzheimer disease • AD brains show characteristic amyloid plaques – insoluble Aβ peptides and hyperposphorylated tau protein • PP2A is a major tau phosphatase • Downregulation may be due to upregulation of PP2A inhibitors

18. References • OMIM • GeneTests • Prosite http://www.expasy.org/prosite/ • ‘Pharmacodynamic Monitoring of Calcineurin Phosphatase Activity in Transplant Patients Treated with Calcineurin Inhibitors’ Yano Drug Metab. Pharmacokinet. 23 (3): 150–157 (2008). • ‘Protein tyrosine phosphatase function: the substrate perspective’ Tiganis and Bennett Biochem. J. (2007) 402, 1–15 • ‘Noonan syndrome’ van der Burgt Orphanet Journal of Rare Diseases 2007, 2:4 • ‘Leopard Syndrome’ Sarkozy et al Orphanet Journal of Rare Diseases 2008, 3:13 • ‘Noonan syndrome and related disorders: Alterations in growth and puberty’ Noonan Rev Endocr Metab Disord (2006) 7:251–255 • ‘Noonan syndrome and related disorders: dysregulated RAS-mitogen activated protein kinase signal transduction.’ Gelb and Tartaglia Hum Mol Genet. 2006 Oct 15;15 • ‘Protein tyrosine phosphatases in the JAK/STAT pathway’ Xu and Qu Front Biosci. ; 13: 4925–4932. • ‘The decision to enter mitosis: feedback and redundancy in the mitotic entry network’ Lindqvist et al J. Cell Biol. Vol. 185 No. 2 193–202 • ‘Aberrant phosphorylation in the pathogenesis of Alzheimer’s disease’ Chung BMB reports