Receptor type protein tyrosine phosphatases (RPTPs) – roles in signal transduction and human disease

Protein tyrosine phosphorylation is a fundamental regulatory mechanism controlling cell proliferation, differentiation, communication, and adhesion. Disruption of this key regulatory mechanism contributes to a variety of human diseases including cancer, diabetes, and auto-immune diseases. Net protein tyrosine phosphorylation is determined by the dynamic balance of the activity of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Mammals express many distinct PTKs and PTPs. Both of these families can be sub-divided into non-receptor and receptor subtypes. Receptor protein tyrosine kinases (RPTKs) comprise a large family of cell surface proteins that initiate intracellular tyrosine phosphorylation-dependent signal transduction in response to binding of extracellular ligands, such as growth factors and cytokines. Receptor-type protein tyrosine phosphatases (RPTPs) are enzymatic and functional counterparts of RPTKs. RPTPs are a family of integral cell surface proteins that possess intracellular PTP activity, and extracellular domains that have sequence homology to cell adhesion molecules. In comparison to extensively studied RPTKs, much less is known about RPTPs, especially regarding their substrate specificities, regulatory mechanisms, biological functions, and their roles in human diseases. Based on the structure of their extracellular domains, the RPTP family can be grouped into eight sub-families. This article will review one representative member from each RPTP sub-family.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

α-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with α-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of α-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that α-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

Involvement of protein tyrosine phosphatases in adipogenesis: New anti-obesity targets?

Obesity is a worldwide epidemic as well as being a major risk factor for diabetes, cardiovascular diseases and several types of cancers. Obesity is mainly due to the overgrowth of adipose tissue arising from an imbalance between energy intake and energy expenditure. Adipose tissue, primarily composed of adipocytes, plays a key role in maintaining whole body energy homeostasis. In view of the treatment of obesity and obesity-related diseases, it is critical to understand the detailed signal transduction mechanisms of adipogenic differentiation. Adipogenic differentiation is tightly regulated by many key signal cascades, including insulin signaling. These signal cascades generally transfer or amplify the signal by using serial tyrosine phosphorylations. Thus, protein tyrosine kinases and protein tyrosine phosphatases are closely related to adipogenic differentiation. Compared to protein tyrosine kinases, protein tyrosine phosphatases have received little attention in adipogenic differentiation. This review aims to highlight the involvement of protein tyrosine phosphatases in adipogenic differentiation and the possibility of protein tyrosine phosphatases as drugs to target obesity. [BMB Reports 2012; 45(12): 700-706]     

Differential roles of estrogen receptors α and β in control of B-cell maturation and selection

It is clear that estrogen can accelerate and exacerbate disease in some lupus-prone mouse strains. It also appears that estrogen can contribute to disease onset or flare in a subset of patients with lupus. We have previously shown estrogen alters B-cell development to decrease lymphopoiesis and increase the frequency of marginal zone B cells. Furthermore, estrogen diminishes B-cell receptor signaling and allows for the increased survival of high-affinity DNA-reactive B cells. Here, we analyze the contribution of estrogen receptor α or β engagement to the altered B-cell maturation and selection mediated by increased exposure to estrogen. We demonstrate that engagement of either estrogen receptor α or β can alter B-cell maturation, but only engagement of estrogen receptor α is a trigger for autoimmunity. Thus, maturation and selection are regulated differentially by estrogen. These observations have therapeutic implications.     

Evolution of the Integrin α and β Protein Families

A phylogenetic analysis of vertebrate and invertebrate α integrins supported the hypothesis that two major families of vertebrate α integrins originated prior to the divergence of deuterostomes and protostomes. These two families include, respectively, the αPS1 and αPS2 integrins of Drosophila melanogaster, and each family has duplicated repeatedly in vertebrates but not in Drosophila. In contrast, a third family (including αPS3) has duplicated in Drosophila but is absent from vertebrates. Vertebrate αPS1 and αPS2 family members are found on human chromosomes 2, 12, and 17. Linkage of these family members may have been conserved since prior to the origin of vertebrates, and the two genes duplicated simultaneously. A phylogenetic analysis of β integrins did not clearly resolve whether vertebrate β integrin genes duplicated prior to the origin of vertebrates, although it suggested that at least the gene encoding vertebrate β4 may have done so. In general, the phylogeny of neither α nor β integrins showed a close correspondence with patterns of α–β heterodimer formation or other functional characteristics. One major exception to this trend involved αL, αM, αX, and αD, a monophyletic group of immune cell-expressed α integrins, which share a number of common functional characteristics and have evolved in coordinated fashion with their β integrin partners. Received: 22 June 2000 / Accepted: 11 September 2000     

Protein tyrosine phosphatase 1B inhibits adipocyte differentiation and mediates TNFα action in obesity

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of systemic glucose and insulin homeostasis; however, its exact role in adipocytes is poorly understood. This study was to elucidate the role of PTP1B in adipocyte differentiation and its implication in obesity. During differentiation of 3T3-L1 white preadipocytes, PTP1B decreased progressively with adipocyte maturation. Lentivirus-mediated PTP1B overexpression in preadipocytes delayed adipocyte differentiation, shown as lack of mature adipocytes, low level of lipid accumulation, and down-regulation of main markers (PPARγ2, SREBP-1c, FAS and LPL). In contrast, lentivirus-mediated PTP1B knockdown accelerated adipocyte differentiation, demonstrated as full of mature adipocytes, high level of lipid accumulation, and up-regulation of main markers. Dominant-negative inhibition on endogenous PTP1B by lentivirus-mediated overexpression of PTP1B double mutant in Tyr-46 and Asp-181 residues (LV-D/A-Y/F) also stimulated adipogenesis, more efficient than PTP1B knockdown. Diet-induced obesity mice exhibited an up-regulation of PTP1B and TNFα accompanied by a down-regulation of PPARγ2 in white adipose tissue. TNFα recombinant protein impeded PTP1B reduction and inhibited adipocyte differentiation in vitro; this inhibitory effect was prevented by LV-D/A-Y/F. Moreover, PTP1B inhibitor treatment improved adipogenesis and suppressed TNFα in adipose tissue of obese mice. All together, PTP1B negatively regulates adipocyte development and may mediate TNFα action to impair adipocyte differentiation in obesity. Our study provides novel evidence for the importance of PTP1B in obesity and for the potential application of PTP1B inhibitors.     

Reciprocal roles of DBC1 and SIRT1 in regulating estrogen receptor α activity and co-activator synergy

Estrogen receptor α (ERα) plays critical roles in development and progression of breast cancer. Because ERα activity is strictly dependent upon the interaction with coregulators, coregulators are also believed to contribute to breast tumorigenesis. Cell Cycle and Apoptosis Regulator 1 (CCAR1) is an important co-activator for estrogen-induced gene expression and estrogen-dependent growth of breast cancer cells. Here, we identified Deleted in Breast Cancer 1 (DBC1) as a CCAR1 binding protein. DBC1 was recently shown to function as a negative regulator of the NAD-dependent protein deacetylase SIRT1. DBC1 associates directly with ERα and cooperates synergistically with CCAR1 to enhance ERα function. DBC1 is required for estrogen-induced expression of a subset of ERα target genes as well as breast cancer cell proliferation and for estrogen-induced recruitment of ERα to the target promoters in a gene-specific manner. The mechanism of DBC1 action involves inhibition of SIRT1 interaction with ERα and of SIRT1-mediated deacetylation of ERα. SIRT1 also represses the co-activator synergy between DBC1 and CCAR1 by binding to DBC1 and disrupting its interaction with CCAR1. Our results indicate that DBC1 and SIRT1 play reciprocal roles as major regulators of ERα activity, by regulating DNA binding by ERα and by regulating co-activator synergy.     

Protein phosphatases and DNA tumor viruses: transformation through the back door?

Cellular transformation by many oncogenic viruses is mediated by alterations in signal transduction pathways that control normal growth and proliferation. Common targets for many transforming viruses are pathways regulated by protein phosphorylation. The biochemical control of proteins in these pathways is a dynamic process that is regulated by the relative activities of protein kinases and phosphatases. Although there are numerous examples of viral oncogenes that encode protein kinases (Hunter, 1991), until recently there has been no evidence linking altered phosphatase activity to transformation. In this review we describe a novel mechanism, utilized by small DNA tumor viruses, in which viral oncogenes bind to and regulate a cellular protein serine/threonine phosphatase. The currently available evidence indicates that alteration of phosphatase activity and subsequent changes in phosphorylation levels is an important step in transformation by these viruses.     

Exploration of biguanido–oxovanadium complexes as potent and selective inhibitors of protein tyrosine phosphatases

The inhibitory effects of three biguanido-oxovanadium complexes ([VO(L(1-3))(2)]·nH(2)O: HL(1) = metformin, HL(2) = phenformin, HL(3) = moroxydine) against four protein tyrosine phosphatases (PTPs) and an alkaline phosphatase (ALP) were investigated. The complexes display strong inhibition against PTP1B and TCPTP (IC(50), 80-160 nM), a bit weaker inhibition against HePTP (IC(50), 190-410 nM) and SHP-1(IC(50), 0.8-3.3 μM) and much weaker inhibition against ALP (IC(50), 17-35 μM). Complex 3 is about twofold less potent against PTP1B, TCPTP and HePTP than complexes 1 and 2, while complex 2 inhibits SHP-1 more strongly (about three to fourfold) than the other two complexes. These results suggest that the structures of the ligands slightly influence the potency and selectivity against PTPs. The complexes inhibit PTP1B and ALP with a typical competitive type.     

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

Protein Kinase C ε Expression in Platelets from Patients with Acute Myocardial Infarction

Objective

Platelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease.

Methods and Results

We therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen.

Conclusions

Our data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.     

GINS Inactivation Phenotypes Reveal Two Pathways for Chromatin Association of Replicative α and ε DNA Polymerases in Fission Yeast

The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of β-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase ε, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase α is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase ε chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase α.     

Overlapping Functions for Two G Protein α Subunits in Neurospora Crassa

Heterotrimeric G proteins, consisting of α, β and γ subunits, mediate a variety of signaling pathways in eukaryotes. We have previously identified two genes, gna-1 and gna-2, that encode G protein α subunits in the filamentous fungus Neurospora crassa. Mutation of gna-1 results in female infertility and sensitivity to hyperosmotic media. In this study, we investigate the expression and functions of gna-2. Results from Western analysis and measurements of gna-2 promoter-lacZ fusion activity indicate that gna-2 is expressed during the vegetative and sexual cycle of N. crassa in both A and a mating types. Activating mutations predicted to abolish the GTPase activity of GNA-2 cause subtle defects in aerial hyphae formation and conidial germination. Extensive phenotypic analysis of Δgna-2 strains did not reveal abnormalities during vegetative or sexual development. In contrast, deletion of gna-2 in a Δgna-1 strain accentuates the Δgna-1 phenotypes. Δgna-1 Δgna-2 strains have a slower rate of hyphal apical extension than Δgna-1 strains on hyperosmotic media. Moreover, Δgna-1 Δgna-2 mutants have more pronounced defects in female fertility than Δgna-1 strains. We propose that gna-1 and gna-2 have overlapping functions and may constitute a gene family. This is the first report of G protein α subunits with overlapping functions in eukaryotic microbes.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Redundant and nonredundant functions of ATM and H2AX in αβ T-lineage lymphocytes

The ataxia telangiectasia mutated (ATM) kinase and H2AX histone tumor suppressor proteins are each critical for maintenance of cellular genomic stability and suppression of lymphomas harboring clonal translocations. ATM is the predominant kinase that phosphorylates H2AX in chromatin around DNA double-strand breaks, including along lymphocyte Ag receptor loci cleaved during V(D)J recombination. However, combined germline inactivation of Atm and H2ax in mice causes early embryonic lethality associated with substantial cellular genomic instability, indicating that ATM and H2AX exhibit nonredundant functions in embryonic cells. To evaluate potential nonredundant roles of ATM and H2AX in somatic cells, we generated and analyzed Atm-deficient mice with conditional deletion of H2ax in αβ T-lineage lymphocytes. Combined Atm/H2ax inactivation starting in early-stage CD4(-)/CD8(-) thymocytes resulted in lower numbers of later-stage CD4(+)/CD8(+) thymocytes, but led to no discernible V(D)J recombination defect in G1 phase cells beyond that observed in Atm-deficient cells. H2ax deletion in Atm-deficient thymocytes also did not affect the incidence or mortality of mice from thymic lymphomas with clonal chromosome 14 (TCRα/δ) translocations. Yet, in vitro-stimulated Atm/H2ax-deficient splenic αβ T cells exhibited a higher frequency of genomic instability, including radial chromosome translocations and TCRβ translocations, compared with cells lacking Atm or H2ax. Collectively, our data demonstrate that both redundant and nonredundant functions of ATM and H2AX are required for normal recombination of TCR loci, proliferative expansion of developing thymocytes, and maintenance of genomic stability in cycling αβ T-lineage cells.     

Mapping of the Mouse Actin Capping Protein α Subunit Genes and Pseudogenes

Capping protein (CP), a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filamentsin vitroand controls actin assembly and cell motilityin vivo.Vertebrates have three α isoforms (α1, α2, α3) produced from different genes, whereas lower organisms have only one gene and one isoform. We isolated genomic clones corresponding to the α subunits of mouse CP and found three α1 genes, two of which are pseudogenes, and a single gene for both α2 and α3. Their chromosomal locations were identified by interspecies backcross mapping. The α1 gene (Cappa1) mapped to Chromosome 3 betweenD3Mit11andD3Mit13.The α1 pseudogenes (Cappa1-ps1andCappa1-ps2) mapped to Chromosomes 1 and 9, respectively. The α2 gene (Cappa2) mapped to Chromosome 6 nearPtn.The α3 gene (Cappa3) also mapped to Chromosome 6, approximately 68 cM distal fromCappa2nearKras2.One mouse mutation,de,maps in the vicinity of the α1 gene. No known mouse mutations map to regions near the α2 or α3 genes.     

Substrate specificity of protein tyrosine phosphatases 1B, RPTPα, SHP-1, and SHP-2

We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately 2 orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active toward multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY-1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY-1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme's in vivo substrate specificity.     

DNA polymerases β and λ and their roles in cell

Among the set of mammalian DNA polymerases, DNA polymerases belonging to the X and Y families have a special place. The majority of these enzymes are involved in repair, including base excision repair and non-homologous end joining. Some of them play a crucial role during the specific process which is referred to as translesion synthesis (TLS). TLS intends for the cell surviving during the replication of damaged DNA templates. Additionally, specific activities of TLS-polymerases have to be useful for repair of double-stranded clustered lesions: if the synthesis is proceeded via base excision repair process, the role of DNA polymerases β or λ will be important. In this review we discussed the biochemical properties and functional relevance of X family DNA polymerases β and λ.