Protein phosphatases

1. Protein phosphatases RCPath Session 4 (pm): Protein motifs 9th December 2008 Stephanie Batey

2. Protein phosphatases • Protein phosphatases = enzymes that catalyze the removal of a phosphate group from the hydroxylated amino acid residue of a protein. • Hydrolysis of phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group. • Directly opposite action to protein kinases: phosphorylation of a given protein is determined by the coordinate control of both protein kinases and phosphatases:

3. Classification of PPases • 2 main groups: - cysteine-dependent phosphatases (CDPs) - metallo-phosphatases (active sites dependent on presence of metal ions for activity). • Protein phosphatases evolved in separate families that are structurally and mechanically distinct • Protein phosphatases can be classified into 3 groups on the basis of sequence, structure and catalytic mechanisms. 1) Ser/ Thr phosphatases 2) Protein Tyr phosphatase (PTPs). Includes dual specificity PPases. 3) Asp-based protein phosphatases with DXDXT/V catalytic subunit. • Classes of protein phosphatases are encoded by 5 gene families.

4. PPase gene families Moorhead et al (2007) Nature 8

5. Serine/ threonine phosphatases • Exist as a single catalytic subunit that binds one or more regulatory domains to form holoenzyme complexes • 2 sub-families of Ser/ Thr PPases: 1) PPP (phosphoprotein phosphatase)- largest family 2) PPM family (protein phophatase Mn2+/Mg2+-dependent). • Catalytic domains are highly conserved. Active site sequence: DXH(X)nDXXD(X)nNH(D/E) • Phosphate removal facilitated by 2 metal ions at the active centre of the enzyme. • Limited number of catalytic subunits in humans (<40) • Specificity, with the exception of PP2C, is defined by additional regulatory or interacting proteins that bind the catalytic subunits, target them to specific locations and/ or substrates and control their activity.

6. Protein tyrosine phosphatases (PTPases) • Encoded by largest family of genes: PTP super family • Single chain multi-domain enzymes: contain ≥ 1 additional motif/ domain outside of catalytic domain • Additional non-catalytic regulatory and targeting domains at both the N- and C-termini are thought to define the function of PTPases. • 104/ 147 human PPase catalytic subunits belong to PTP family. • Stricter specificity and tighter regulation compared to Ser/ Thr PPases • Each PTPase contains highly conserved catalytic domain of ~240 residues: defined by active-site signature motif of 10 residues HCSAGxGRxG/ Cys-X5-Arg • Cysteine residue critical → functions as nucleophile which binds phosphate Arginine residue → binds to oxygen atoms of the phosphate

7. Protein tyrosine phosphatases (2) • Subfamilies include classic PTPases dual specificity phosphatases Classic PTPases • Depth of active site cleft renders the enzyme specific for phosphorylated Tyr residues only • Subdivided into: - Transmembrane receptor-like enzymes - Soluble cytosolic enzymes • Most receptor-like PTPs have 2 intracellular PTP domains: N-terminal domain accounts for most of catalytic activity. C-terminal domain is needed for physiological function. • Ligand-induced dimerization of 2 PTP domains may inhibit phosphatase activity.

8. Dual specificity phosphatases (DSPases) • Approximately 65 genes encode DSPases • Share the conserved PTPase active site motif and use the same catalytic mechanism but unique in their ability to dephosphorylate Tyr, Ser or Thr, phosphoinositides or mRNA (due to shallower active site cleft) • Not dependent on recognition of phosphorylated residue for substrate binding. Bind through separate binding domain. Docking interactions regulate the specificity of DSPs. • Key role in the dephosphorylation of MAP kinases (also termed MAP kinase phosphatases). • All DSPs share strong amino-acid sequence homology in their catalytic domains. DX26(V/L)X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M, where X can be any amino acid. • Catalytically important aspartic acid located on flexible loop. When substrate binds enzyme → conformational change → moves aspartic acid into active site.

9. Schematic of PPases

10. Regulation of PPases • Regulation of Ser/ Thr PPases and PTPs similar: via protein-protein and protein-phospholipid interactions. • Targeting to substrate-containing locations or complexes and direct allosteric modulation of the catalytic domain/ subunit important. • Targets include enzymes (eg. glycogen synthase), transcription factors (eg. CREB), RNA splicing factors, cell surface receptors, cytoskeletal proteins, channels (eg, Ca2+ release channels), pumps and ATPases. • Recent data: phophorylation preferentially targets nuclear proteins

11. Regulatory pathways • Phosphorylation and dephosphorylation of structural and regulatory proteins are major intracellular control mechanisms in eukaryotes. - alter activity, location and stability of the target protein. • Protein dephosphorylation is a key process involved in cell signalling: PPases provide fine control by regulating the rate and duration of a signalling response (protein kinases thought to control the amplitude of a signalling response). • Evidence beginning to emerge for specific regulatory roles for protein phosphatases in exocytosis, which is integral to many signal transduction pathways: pathological dysregulation of exocytosis associated with a variety of disorders including epilepsy, hypertension, diabetes and asthma. • PPases important for neuronal development. In neurons, PP1 is highly conserved in dendritic spines. Involved in neuronal signalling.

12. Functions of PPases • Roles for PPases in: • Regulation of cell cycle progression • Regulation of cell growth and apoptosis • Regulation of cell adhesion, motility and cytoskeletal function. • DNA replication and repair • Ribosome biogenesis • Chromatin remodelling. • Spliceosome assembly and catalytic steps of RNA processing. • Transcriptional control.

13. PPases in disease (1) • Loss of PPases reported in cancer: >30 different PTPs and loss may be genetic or epigenetic eg. DUSP6/ MKP-3 • Several PPases found to be over expressed in human malignancies eg. PRL3 in metastatic colon cancer, Cdc25A and B • Also highly expressed in brains of AD patients and may contribute to the pathology of neurodegeneration. • Inherited genetic diseases: - Noonan syndrome (SHP2) - Lafora’s epilepsy (laforin) - Muscle dystrophies (myotubularin) - Immunodeficiencies (CD45) Others: - autoimmune diseases - acute myeloblastic leukaemia - Opitz BBB/G syndrome

14. PPases in disease (2) Type 2 diabetes and obesity • PTPs involved in negative regulation of insulin signalling. • Increased activity of PTPs eg. PTP1B may contribute to insulin resistance in a subset of affected patients Immunodeficiences • Leukocyte common antigen CD45 = abundant transmembrane receptor-like PTP expressed exclusively on haemapoietic cells. • Positive role in promoting signalling through T and B cells. • Transgenic mice with activating CD45 mutations had lymphoproliferation and severe autoimmune disease. • Two SCID patients reported with CD45 mutations and multiple sclerosis may be due to abnormally high CD45 expression levels. Muscle dystrophies • Myotubularin genes encode large family of PPases. hMTM1 mutated myotubular myopathy. • hMTMR2 mutated in recessive forms of Charcot Marie Tooth neuropathy.

15. PPases in disease (3) Lafora disease • Caused by mutations in Laforin/ EPM2A gene which encodes a DSP • Fatal AR disorder – progressive neurodegeneration, myoclonus and epilepsy. • Laforin shown to be involved in regulation of glycogen metabolism. Noonan syndrome • PTPN11 gene encodes a PTP, SHP2, which normally facilitates Ras activation. • Missense mutations in PTPN11 cause Noonan syndrome. Typically cause hyperactivation/ gain of function. • Mutations restricted to the PTP catalytic site cause LEOPARD syndrome. Typically loss of function/ dominant negative. • Activating somatic mutations in PTPN11 associated with an increased risk for some sporadic childhood cancers.

16. Bioinformatics • Website: each PTP locus is hyperlinked to disease information in OMIM, Mitelman and PubMed: http://science.novonordisk.com/ptp or http://ptp.cshl.edu • PROSITE website • Alamut software: PolyPhen, SIFT, Align GVGD, SwissUNIPROT

17. PPases as therapeutic targets? • Aberrant protein phosphorylation linked to many human diseases. Protein kinases are second largest drug target. Protein phosphatases only just being recognised as future therapeutic targets. • Function of certain phosphatase families defined by the regulatory or interacting proteins that bind the catalytic subunits, target to specific locations/ substrates and control their activity. • Identification of regulatory proteins to help define the holoenzyme complexes and develop techniques to disrupt specific phosphatase-substrate interactions.

18. References • Andersen et al (2004) FASEB J. 18: 8-30 • Hendriks et al (2008) FEBS journal 816-830 • Moorhead et al (2007) Nature Reviews (8) • Andersen et al (2005) Methods 35:90-114 – Bioinformatics paper • Sim et al (2003) Biochem. J 373: 641-659 • Mustelin, T. Methods in Molecular Biology 365: Protein Phosphatase Protocols. • Ducruet et al (2005) Annu. Rev. Med. 45: 725-50 • Tonks (2006) Nature Reviews: Vol 7 • Camps et al (1999) FASEB J. 14: 6-16 • PROSITE website.