Protein Motifs

1. Protein Motifs Protein Phosphatases Roy Poh Feb 08

2. Phosphatase • Enzymes that removes a phosphate group from its substrate by hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group (dephosphorylation). • This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their substrates by using energetic molecules like ATP. • Two main groups are: Cysteine-dependent Phosphatases (CDPs) Metallo-phosphatases (which are dependent on metal ions in their active sites for activity)

3. Phosphatases can be subdivided based upon their substrate specificity.

4. Physiological Relevance • Phosphatases act in opposition to kinases/phosphoylases, which add phosphate groups to proteins. The addition of a phosphate group may activate or de-activate an enzyme (e.g., Kinase signalling pathways) or enable a protein-protein interaction to occur (e.g., SH3 domains); therefore phosphatases are integral to many signal transduction pathways. • The addition and removal of phosphate does not necessarily correspond to enzyme activation or inhibition. Enzymes can have separate phosphorylation sites for activating or inhibiting functional regulation. • Protein phosphatases are grouped into 3 distinct classes: tyrosine-specifc, Serine/threonine-specific and dual specificity phosphatase

5. Protein Tyrosine Phosphatase • PTPase catalyze the removal of a phosphate group attached to a tyrosine residue (involving a Cysteine nucleophile and an Arginine residue that binds to oxygen atoms of the phosphate) • These enzymes are very important in the control of cell growth, proliferation, differentiation and transformation. • Multiple forms of PTPase have been characterized and can be classified into 2 groups: Soluble PTPases and Transmembrane receptor proteins that contain PTPase domain • The diversity of PTPases is primarily due to the variety of non-catalytic regulatory sequences and targeting domains attached to both N- and C-terminals. • Protein Tyrosine Phosphatase Non-receptor type 1 (PTPN1) is the founding member of over 40 PTPases.

6. PTPase (2) • PTPases contain a highly conserved catalytic domain of ~240 amino acid residues. • PTPases have a distinctive active site signature motif of 10 residues: HCSAGxGRxG [or C-x(5)-R motif]. • Highly conserved Arginine and Cysteine residues at the catalytic domain are considered to be essential for enzyme activity • The depth of the active site cleft renders the enzyme specific for phosphorylated Tyr (pTyr) residues, instead of pSer or pThr

7. Examples of PTPase Soluble (Non-transmembrane) PTPases • PTPN1 (PTP-1B) – most studied, 37kDa enzyme, a single domain organised into 8 a-helices and 12 b-sheets with a catalytic PTP loop. • PTPN2 (T-cell PTPase; TC-PTP). • PTPN3 (H1) and PTPN4 (MEG), enzymes that contain an N-terminal FERM domain and could act at junctions between the membrane and cytoskeleton. • PTPN5 (STEP). • PTPN6 (PTP-1C; HCP; SHP) and PTPN11 (PTP-2C; SH-PTP3; Syp): enzymes which contain two copies of the SH2 domain at its N-terminal extremity. The Drosophila protein corkscrew (gene csw) also belongs to this subgroup. • PTPN7 (LC-PTP; Hematopoietic protein-tyrosine phosphatase; HePTP).

8. Examples of PTPase (2) Receptor-like (Transmembrane) PTPases • Structurally, all known receptor PTPases, are made up of a variable length extracellular domain, followed by a transmembrane region and a C-terminal catalytic cytoplasmic domain. • Some of the receptor PTPases contain fibronectin type III (FN-III) repeats, immunoglobulin-like domains, MAM domains or carbonic anhydrase-like domains in their extracellular region. • The cytoplasmic region generally contains two copies of the PTPase domain. • The first seems to have enzymatic activity, while the second is inactive but seems to affect substrate specificity of the first. • In these domains, the catalytic cysteine is generally conserved but some other, presumably important, residues are not.

9. Examples of PTPase (3) Dual specificity PTPases • DSP1 (PTPN10; MAP kinase phosphatase-1; MKP-1); which dephosphorylates MAP kinase on both Thr-183 and Tyr-185. • DSP2 (PAC-1), a nuclear enzyme that dephosphorylates MAP kinases ERK1 and ERK2 on both Thr and Tyr residues. • DSP3 (VHR) • DSP4 (HVH2) • DSP5 (HVH3) • DSP6 (Pyst1; MKP-3). • DSP7 (Pyst2; MKP-X).

10. PTPase domain • PTPase domains consist of about 300 amino acids. There are two conserved cysteines, the second one has been shown to be absolutely required for activity. Furthermore, a number of conserved residues in its immediate vicinity have also been shown to be important. • A signature pattern for PTPase domains centred on the active site cysteine. • There are three profiles for PTPases, the first one spans a short region that is common to both dual-specificity protein phosphatases and PTPases. The second and third ones cover the whole domain and are respectively specific for Ser/Thr and Tyr protein phosphatases, and Tyr protein phosphatases.

11. PTPN11 • PTPN11 possesses a domain structure that consists of two tandem SH2 (Src homology-2) domains in its N-terminus followed by a protein tyrosine phosphatase PTP domain • In the inactive state, the N-terminal SH2 domain binds the PTP domain and blocks access of potential substrates to the active site. • Upon binding to target phospho-tyrosyl residues, the N-terminal SH2 domain is released from the PTP domain, catalytically activating the enzyme by relieving this auto-inhibition • Mutations in PTPN11 are responsible for Noonan and LEOPARD syndrome and also seen in various leukaemias (JMML, AML, CMML). • In Noonan, mutations are mostly in the binding interface between the N-SH2 domain and the PTP catalytic core and shown to cause hyper-activation (gain of function). • In LEOPARD, mutations appeared to be more specific and are restricted to the PTP catalytic core and loss of function/dominant negative have been suggested.

12. Comparison of PTPN11 mutations causing Noonan syndrome and cancers. (A) Location of mutated residues in the three-dimensional structure of SHP-2 in its catalytically inactive conformation (green, N-SH2 domain; cyan, C-SH2 domain; pink, PTP domain). Residues altered in Noonan syndrome (left) or leukemias (right) are shown with their lateral chains colored according to the proposed classification (red, group I; yellow, group II; green, group III; cyan, group IV; orange, group V; violet, group VI; blue, unclassified) (3).. (B) Distribution of SHP-2 mutations in human disease. mutations and their relative prevalence in hematologic malignancies (above), and Noonan syndrome and related developmental disorders (below). JMML: juvenile myelomonocytic leukemia; ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; CMML: chronic myelomonocytic leukemia; MDS: myelodysplastic syndromes.

13. Ser/Thr protein phosphatase • Enzymes that remove the serine- / threonine-bound phosphate group from a wide range of phospho-proteins • Four major classes have been described: PP1, PP2A, PP2B, and PP2C • Differ in their substrate specificity, divalent cation requirements, and sensitivity to inhibitors • Type I phosphatases, which include PP1, can be inhibited by two heat-stable proteins known as Inhibitor-1 (I-1) and Inhibitor-2 (I-2). They preferentially dephosphorylate the b-subunit of phosphorylase kinase. • Type II phosphatases are insensitive to heat-stable inhibitors and preferentially dephosphorylate the a-subunit of phosphorylase kinase. Type II phosphatases are subdivided into spontaneously active (PP2A), Ca2+-dependent (PP2B), and Mg2+-dependent (PP2C) classes of phosphatases. • PP1, PP2A, and PP2B phosphatases have catalytic subunits with significant sequence similarity, and regulatory subunits which are believed to regulate activity or cellular localization.

14. Ser/Thr protein phosphatase • PP2C phosphatases are monomeric and have amino acid sequences that are distinct from those of the other phosphatases. However it displays some degree of overlapping substrate specificity with PP1 & PP2A. • Multiple PP2C isozymes recognised in human. • Consensus pattern: • [LIVMFY]-[LIVMFYA]-[GSAC]-[LIVM]-[FYC]-D-G-H-[GAV] (The D is a Manganese ligand) • Protein phosphatase magnesium-dependent 1 gama isoform, PPM1G (PP2-gamma) contains highly conserved sequence of PP2C family. PPM1G shared only 34% identity to human PP2C-alpha. A distinctive feature of PPM1G is the presence of a large acidic domain.

15. Dual Specificity phosphatase • DSPs play a key role in the dephosphorylation of MAP kinases. Hence, they are also termed as MAP kinase phosphatases (MKPs) • On the basis of structures, predicted from genomic sequence, MKPs have been divided into three subgroups. Group I - DSP1, 2, 4, and 5 Group II - DSP6, 7, 9 and 10 Group III - DSP8 and 16. • All the DSPs share strong amino-acid sequence homology in their catalytic domains. The catalytic domain contains a highly conserved consensus sequence: D X 26(V/L)X(V/I)HC XAG(I/V)SR SXT(I/V)XXAY(L/I)M • D, C & R reported to be essential for the catalytic activity of DSPs

16. DSP • The cysteine is required for the nucleophilic attack on the phosphorus of the substrate and the formation of the thiol-phosphate intermediate. • The conserved arginine binds the phosphate group of the phospho-tyrosine or phospho-threonine, enabling transition-state stabilization; • The aspartate enhances catalysis by protonating oxygen on the departing group (tyrosine or threonine) • All DSPs contain two conserved regions, known as the CH2 domains, at their amino-terminus, which are believed to be involved in substrate binding • In addition, a MAP kinase-docking site at their amino-terminus, which consists of a cluster of positively charged amino acids. • The corresponding docking site on the MAP kinases is shown to consist of negatively charged residues indicating that electrostatic interactions are important for the binding of MAP kinases and MKPs.

17. DSP • The group III DSPs also have an extended carboxyl terminus containing PEST sequences [abundant in proline (P), glutamate (E), serine (S) and threonine (T) residues] which are normally found in rapidly degrading proteins. • Removal of the PEST sequences from these proteins can result in their stabilization. • Most DSPs display wide tissue distribution, however, some exhibit a tissue-specific expression pattern. • For example, DSP8 mainly in brain, heart, and lung; DSP9 in placenta and kidney; and DSP10 is seen only in liver and skeletal muscle. • The intracellular distribution of DSPs is also variable

18. Source • Websites: • OMIM • Prosite • Protein tyrosine phosphatases and signalling. Stoke AW. Journal of Endocrinology (2005) 185, 19-33 • Noonan syndrome and related disorders: dysregulated RAS-mitogen activated protein kinase signal transduction. Bruce D. Gelb1, and Marco Tartaglia. Hum Mol Gen (2006) 15, 220-226 • PTPN11 muations in LEOPARD sydrome have dominant negative, not activating effects. Kontaridis M et al. J Biol Chem (2006) 281, 6785-6792