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PTP 101 - Introduction to Protein Tyrosine Phosphatases

Introduction
The reversible phosphorylation of proteins as carried out by protein kinases and phosphatases is one of the most widespread mechanisms for controlling cellular functions (1): cells can quickly respond to intracellular and extracellular cues by altering the phosphorylation status of target proteins with the effects of increasing or decreasing their biological activity, modifying their sub-cellular localisation, or affecting protein stability and protein-protein interactions (2). Reversible protein phosphorylation is thus a simple and flexible regulatory system that has been positively selected for in evolution as a general mechanism of cellular control. The first serine/threonine protein kinase (‘Phosphorylase Kinase’) was reported by Fischer and Krebs as early as 1955 (3, 4). It took another 25 years to realize that v-Src (encoded by the Rous sarcoma virus) was a protein kinase (5) that phosphorylates tyrosine residues (a PTK, (6)). On the other hand, the first serine/threonine protein phosphatases were discovered during the late 1970’s and early 1980’s (7), and the first tyrosine-specific phosphatase (PTP1B) in 1988 (8).

Tyrosine phosphorylation in metazoans is a fundamental signaling mechanism controlling a plethora of processes ranging from development to cellular shape and motility, transcriptional regulation, and proliferation vs. differentiation decisions. Not surprisingly, the abnormal regulation of tyrosine phosphorylation on target proteins is responsible for a wide spectrum of human conditions, including diabetes, obesity, cancer, and inflammatory diseases. Many diseases have been associated to PTKs as well as to protein tyrosine phosphatase (PTP) over-expression and deficiencies (9).

Historically, research on PTKs has advanced at a faster rate than investigations into PTPs. Not only were PTKs identified nearly a decade earlier than PTPs, but also the intrinsic difficulties of investigating the “disappearance” of a phosphate moiety as opposed to the appearance of the radioactive phosphate represented a major burden for the PTP field. Yet the use of both embryonic gene targeting models, and the knockdown technologies of siRNA and shRNA have validated the importance of PTP activities in a great number of signaling pathways. Moreover, major advances have been made with the development of substrate trapping techniques where specific mutations of the PTP catalytic domains allow the purification, detection and identification of their physiological substrates (10).

The human genome contains 109 PTP-coding genes with the characteristic PTP signature motif (C(X)5R), and are split into the following families (11):

Class I
Class I are the largest group of PTPs and catalytically are cysteine-based enzymes, including the classical tyrosine-specific phosphatases (both receptor and cytoplasmic), and the dual-specificity phosphatases (DSPs, or VH1-like). DSPs represent the most promiscuous group of PTPs in terms of substrate specificity, with some members dephosphorylating mRNAs while other enzymes dephosphorylate lipids. DSPs can be subdivided into 7 distinct groups based on their domains and sequences: Atypical phosphatases, Myotubularins, MKPs, PRLs, PTENs, CDC14s and Slingshots. PTENs and Myotubularins are lipid phosphatases targeting inositol triphosphates that are D3 phosphorylated. In human, each of the 15 myotubularin enzymes (genes) specifically targets different types of lipid substrates. In addition, PTEN can also dephosphorylate certain myotubularins. Atypical phosphatases are a large group of DSPs targeting an array of substrates, although some members are catalytically inactive and act as scaffold proteins. Slingshot phosphatases were first identified in Drosophila where they function in eye development. Slingshots inhibit actin polymerization by targeting cofilin and the actin-depolymerization factor. These phosphatases play roles in wound healing and can be regulated by protein kinase D. The PRL phosphatases are characterized by the presence of a CAAX prenylation motif at the C-terminus, which means that the farnesylated enzymes are targeted to the cell membrane and the inner membrane. CDC14 enzymes play important roles in DNA repair, DNA damage and the cell cycle. CDC14 PTPs primarily target proteins that have been phosphorylated by proline-dependent kinases. MKPs or mitogen-activated protein kinase phosphatases act on MAP kinases.

Class II
Class II PTPs are a small but evolutionarily conserved class of PTPs with only one member in human (ACP1); they are also found in bacteria and are structurally related to bacterial arsenate reductases. Class II PTPs specifically target phosphorylated tyrosine residues.

Class III
Class III PTPs, like the Class I and II members, are also cysteine-based enzymes displaying specificity towards phosphotyrosine and phosphothreonine residues. The human enzymes (CDC25A, CDC25B and CDC25C) control cell cycle progression by dephosphorylating cyclin-dependent kinases. Despite sharing a cysteine-based catalytic mechanism, Class I, II, and III PTPs are believed to have evolved independently.

Class IV
A fourth class of PTPs displays an aspartic acid-based catalytic mechanism with dependence on a cation, and is represented by the developmentally important EyA (‘Eyes Absent’) genes, of which only 1 member is found in the fruit fly versus 4 genes in mouse and human. EyA enzymes target phosphotyrosines as well as phosphoserine residues, and EyA substrates include proteins, phospholipids and nucleotides. EyA phosphatases play critical developmental roles, and also function in epigenetic regulation by targeting phosphorylated histones.

References

(1) Cohen P (2002) The origins of protein phosphorylation. Nat Cell Biol, 4:E127-130. PMID: 11988757

(2) Cohen P (2000) The regulation of protein function by multisite phosphorylation--a 25 year update. Trends Biochem Sci. 25:596-601. PMID: 11116185

(3) Fischer EH, Krebs EG (1955) Conversion of phosphorylase b to phosphorylase a in muscle extracts. J Biol Chem. 216:121-132. PMID: 13252012

(4) Krebs EG, Fischer EH (1956) The phosphorylase b to a converting enzyme of rabbit skeletal muscle. Biochim Biophys Acta 20:150-157. PMID: 13315361

(5) Collett MS, Erikson RL (1978) Protein kinase activity associated with the avian sarcoma virus src gene product. Proc Natl Acad Sci USA 75:2021-2024. PMID: 205879

(6) Hunter T, Sefton BM (1980) Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A 77:1311-1315. PMID: 6246487

(7) Ingebritsen TS, P. Cohen (1983) The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities. Eur J Biochem. 132:255-261. PMID: 6301824

(8) Tonks NK, Diltz CD, Fischer EH (1988) Purification of the major protein-tyrosine-phosphatases of human placenta. J Biol Chem. 263:6722-6730. PMID: 2834386

(9) Hendriks WJ, Elson A, Harroch S, Pulido R, Stoker A, den Hertog J (2013) Protein tyrosine phosphatases in health and disease. FEBS J 280(2):708-30. PMID: 22938156

(10) Tonks NK (2013) Protein tyrosine phosphatases--from housekeeping enzymes to master regulators of signal transduction. FEBS J. 280(2):346-78. PMID: 23176256

(11) Alonso A, Sasin J, Bottini N, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T (2004) Protein tyrosine phosphatases in the human genome. Cell 117:699-711. PMID: 15186772


Examples of well-studied PTPs

PTP1B
PTP1B (PTPN1 gene) was the first non-receptor tyrosine phosphatase to be discovered, and much of what is known about basic phosphatase functionality is derived from studies on this archetypal PTP. It is a ubiquitously expressed enzyme with complex regulation and mechanisms of action, and as such has been associated with a range of diseases from cancer to metabolism, immunity and cardiovascular disease (1-3). PTP1B inactivates the insulin receptor and thus insulin receptor signaling, and negatively modulates leptin receptor signaling (1, 4, 5). PTP1B knockout mice are more sensitive to insulin and leptin, are resistant to obesity when maintained on a high-fat diet in addition to having a better tolerance to glucose (6, 7). Furthermore, PTP1B inhibition resulted in a decrease in heart failure, a protective effect also related to enhanced insulin receptor signaling upon PTP1B inhibition (3). PTP1B has been shown to activate the ERK, PDGF and IGF growth factor receptors as well as Src family kinases (8, 9). It is now also recognized as a player in many cancers, displaying both tumor-promoting and suppressive effects (5, 8-10).

PTPN11

Several diseases are associated with mutations in PTPN11, the gene encoding Src homology 2 domain-containing protein-tyrosine phosphatase (Shp-2). Shp-2 activates the Ras and ERK kinases in several manners, and plays a role in cytokine signaling.  Shp-2 mutations are commonly associated with developmental disorders and cancer. These include the Noonan and LEOPARD syndromes (which display common characteristics), as well as cardiac abnormalities, short stature, craniofacial aberrations and susceptibility to cancer (11).  Interestingly, despite their similarities, the Noonan syndrome is associated with loss-of-function mutations, whereas the LEOPARD syndrome is associated with gain-of-function Shp-2 mutations, which are present in different areas of the gene (11, 12). These mutations and the phenotype that manifests in these developmental disorders point to various roles of Shp-2 in heart, immune and CNS development. Furthermore, even patients with activating Shp-2 mutations which do not manifest as developmental syndromes, are predisposed to cancers such as childhood leukemias (11).

PTPN22
Lymphoid tyrosine phosphatase (Lyp), encoded by the PTPN22 gene, is part of the PEST family of phosphatases. Its expression is restricted to hematopoietic cells and is associated with infection, cancer, autoimmunity and metabolism (13). Lyp has been identified as the major risk gene for autoimmunity, after the major histocompatibility complex (14). Genome-wide association studies have traced this back to the arginine to tryptophan substitution R620W (15). This mutation has been shown to convey susceptibility to diseases such as type 1 diabetes, rheumatoid arthritis and systemic lupus erythematosus (16). Studies have shown that Lyp acts as a modulator of T-cell receptor (TCR) signaling by inactivating Src and Syk kinases through association with Csk kinase, all which are key modulators of TCR signaling (14). Moreover, it has been suggested that R620W favors an autoimmune state by promoting pro-inflammatory Th1 responses. However, the R620W enzyme is part of a complex regulatory network that can convey both protective and disease-promoting effects in different contexts, with some studies suggesting that different splice variants may be involved (17, 18). Experimental work as well as the positioning of this gene on a chromosomal area where genomic rearrangements are common, suggest that this PTP may play a yet undefined oncogenic role in hematological malignancies (19).

References

(1) Cho H (2013) Protein tyrosine phosphatase 1B (PTP1B) and obesity. Vitam Horm 91:405-424. PMID: 23374726

(2) Yip SC, Saha S, Chernoff J (2010) PTP1B: a double agent in metabolism and oncogenesis. Trends Biochem Sci. 35:442-449. PMID: 20381358

(3) Gomez E et al (2012) Reduction of heart failure by pharmacological inhibition or gene deletion of protein tyrosine phosphatase 1B. J Mol Cell Cardiol 52:1257-1264. PMID: 22446161

(4) Stuible M et al (2007) Cellular inhibition of protein tyrosine phosphatase 1B by uncharged thioxothiazolidinone derivatives. Chembiochem 8:179-186. PMID: 17191286

(5) Stuible M, Doody KM, Tremblay ML (2008) PTP1B and TC-PTP: regulators of transformation and tumorigenesis. Cancer Metastasis Rev. 27:215-230. PMID: 18236007

(6) Elchebly M et al (1999) Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283:1544-1548. PMID: 10066179

(7) Tiganis T (2013) PTP1B and TCPTP--nonredundant phosphatases in insulin signaling and glucose homeostasis. FEBS J 280:445-458. PMID: 22404968

(8) Julien SG et al (2007) Protein tyrosine phosphatase 1B deficiency or inhibition delays ErbB2-induced mammary tumorigenesis and protects from lung metastasis. Nat Genet. 39:338-346. PMID: 17259984

(9) Lessard L, Stuible M, Tremblay ML (2010) The two faces of PTP1B in cancer. Biochim Biophys Acta 1804:613-619. PMID: 19782770

(10) Lessard L et al (2012) PTP1B is an androgen receptor-regulated phosphatase that promotes the progression of prostate cancer. Cancer Res. 72:1529-1537. PMID: 22282656

(11) Grossmann KS, Rosario M, Birchmeier C, Birchmeier, W (2010) The tyrosine phosphatase Shp2 in development and cancer. Adv Cancer Res. 106:53-89. PMID: 20399956

(12) Yu ZH et al (2013) Structural and Mechanistic Insights into LEOPARD Syndrome-Associated SHP2 Mutations. J Biol Chem. 288(15)10472-82. PMID: 23457302

(13) Stanford, SM, Mustelin TM, Bottini N (2010) Lymphoid tyrosine phosphatase and autoimmunity: human genetics rediscovers tyrosine phosphatases. Semin Immunopathol 32:127-136. PMID: 20204370

(14) Burn GL, Svensson L, Sanchez-Blanco C, Saini M, Cope AP (2011) Why is PTPN22 a good candidate susceptibility gene for autoimmune disease? FEBS Lett 585:3689-3698. PMID: 21515266

(15) Bottini N et al (2004) A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet. 36:337-338. PMID: 15004560

(16) Rhee I, Veillette A (2012) Protein tyrosine phosphatases in lymphocyte activation and autoimmunity. Nat Immunol. 13:439-447. PMID: 22513334

(17) Diaz-Gallo LM, Martin J (2012) PTPN22 splice forms: a new role in rheumatoid arthritis. Genome Med 4:13. PMID: 22364193

(18) Ronninger M et al (2012) The balance of expression of PTPN22 splice forms is significantly different in rheumatoid arthritis patients compared with controls. Genome Med 4:2. PMID: 22264340

(19) Hardy S, Julien SG, Tremblay ML (2012) Impact of oncogenic protein tyrosine phosphatases in cancer. Anticancer Agents Med Chem 12:4-18. PMID: 21707506


Genetic polymorphisms

Approximately 50% of human PTPs have been associated with acquired and hereditary conditions, with research showing specialized roles of PTPs in many diseases. Polymorphisms in PTP genes have been shown to have a great influence on disease susceptibility and phenotype. Point mutations, amplifications and deletions resulting in either gain-of function or loss-of function phenotypes have been observed. Each phosphatase can exert its action on a number of targets and redundant functions of these are evident based on the absence of a strong phenotype in certain PTP knockout mouse models. However, the functions of these are so finely tuned that even a polymorphisms conveying a slight alteration of action can contribute to disease and studies suggest a domineering role of PTPs over protein kinases in the regulation of signaling events (1). Differences in PTP responses and expression signatures also exist across healthy individuals. Therefore, when referring to PTPs in the context of treatment, this point must also be taken into account since such variabilities would likely result in different treatment outcomes across patients (1). Therefore, PTP polymorphisms play important roles in disease. A well-studied example is PTP1B where a number of genetic polymorphisms have been associated with increased susceptibility to some diseases, in addition to affecting the disease outcome. For example, three independent groups showed that the 1484insG plays a role in insulin resistance, one of which demonstrated that this was due to PTP1B overexpression (2-4). On the other hand, the 981CT SNP has been associated with a protective effect against type 2 diabetes. Additional roles for polymorphisms have been found in related disorders such as obesity, hypertension and lipid metabolism (5-10). In line with the polymorphism associations, the 20q13 chromosomal region to which PTP1B has been mapped, is identified as a type 2 diabetes and obesity quantitative trait locus. Moreover, this region is also frequently subject to amplification in breast cancer, although an association with polymorphisms has yet to be made. However, given the role in supporting mammary tumorigenesis in mice, it is possible that there is some genetic variability conveying susceptibility/resistance there as well (11).

References

(1) Tautz L, Pellecchia M, Mustelin T (2006) Targeting the PTPome in human disease. Expert Opin Ther Targets 10:157-177. PMID: 16441235

(2) Di Paola R et al (2002) A variation in 3' UTR of hPTP1B increases specific gene expression and associates with insulin resistance. Am J Hum Genet. 70:806-812. PMID: 11833006

(3) Gu HF et al (2000) Association between the human glycoprotein PC-1 gene and elevated glucose and insulin levels in a paired-sibling analysis. Diabetes 49:1601-1603. PMID: 10969847

(4) Meshkani R et al. (2007) 1484insG polymorphism of the PTPN1 gene is associated with insulin resistance in an Iranian population. Arch Med Res 38: 556-562. PMID: 17560463

(5) Bauer F et al (2010) PTPN1 polymorphisms are associated with total and low-density lipoprotein cholesterol. Eur J Cardiovasc Prev Rehabil. 17:28-34. PMID: 20177231

(6) Ukkola O et al (2005) Protein tyrosine phosphatase 1B variant associated with fat distribution and insulin metabolism. Obes Res. 13:829-834. PMID: 15919835

(7) Olivier M et al (2004) Single nucleotide polymorphisms in protein tyrosine phosphatase 1beta (PTPN1) are associated with essential hypertension and obesity. Hum Mol Genet 13:1885-1892. PMID: 15229188

(8) Kipfer-Coudreau S et al (2004) Single nucleotide polymorphisms of protein tyrosine phosphatase 1B gene are associated with obesity in morbidly obese French subjects. Diabetologia 47:1278-1284. PMID: 15235769

(9) Cheyssac C et al (2006) Analysis of common PTPN1 gene variants in type 2 diabetes, obesity and associated phenotypes in the French population. BMC Med Genet. 7:44. PMID: 16677372

(10) Tsou RC, Bence KK (2012) The Genetics of PTPN1 and Obesity: Insights from Mouse Models of Tissue-Specific PTP1B Deficiency. J Obes. 2012: 926857. PMID: 22811891

(11) Shi H et al (2011) Single nucleotide polymorphisms in the 20q13 amplicon genes in relation to breast cancer risk and clinical outcome. Breast Cancer Res Treat. 130:905-916. PMID: 21630024