Tyrosine phosphatase PTPα contributes to HER2-evoked breast tumor initiation and maintenance.

Protein tyrosine phosphatase alpha (PTPα/PTPRA) was shown previously to be overexpressed in human primary breast cancers, and to suppress apoptosis in estrogen receptor-negative breast cancer cells in vitro. However, it is not known whether PTPα is important for mammary tumor initiation, maintenance and/or progression. We have used a combination of three-dimensional cultures, a transgenic mouse model of breast cancer lacking PTPα as well as xenografts of human breast cancer cell lines to address these questions. We found that PTPα knockdown after overt tumor development reduced the growth of HER2-positive human breast cancer cell lines, and that this effect was accompanied by a reduction in AKT phosphorylation. However, PTPα knockdown did not affect invasiveness of HER2-positive human breast cancer cells grown in three-dimensional cultures. Moreover, in MMTV-NeuNT/PTPα(-/-) mice, PTPα ablation did not affect NeuNT-evoked tumor onset or metastasis but decreased the number of tumors per mouse. Thus, we demonstrate that PTPα contributes to both HER2/Neu-mediated mammary tumor initiation and maintenance. Our results suggest that inhibition of PTPα can have a beneficial effect on HER2-positive breast cancers, but that inhibition of additional targets is needed to block breast tumorigenesis.

Distinct FAK-Src activation events promote alpha5beta1 and alpha4beta1 integrin-stimulated neuroblastoma cell motility.

Signals from fibronectin-binding integrins promote neural crest cell motility during development in part through protein-tyrosine kinase (PTK) activation. Neuroblastoma (NB) is a neural crest malignancy with high metastatic potential. We find that alpha4 and alpha5 integrins are present in late-stage NB tumors and cell lines derived thereof. To determine the signaling connections promoting either alpha4beta1- or alpha5beta1-initiated NB cell motility, pharmacological, dominant negative and short-hairpin RNA (shRNA) inhibitory approaches were undertaken. shRNA knockdown revealed that alpha5beta1-stimulated NB motility is dependent upon focal adhesion kinase (FAK) PTK, Src PTK and p130Cas adapter protein expression. Cell reconstitution showed that FAK catalytic activity is required for alpha5beta1-stimulated Src activation in part through direct FAK phosphorylation of Src at Tyr-418. Alternatively, alpha4beta1-stimulated NB cell motility is dependent upon Src and p130Cas but FAK is not essential. Catalytically inactive receptor protein-tyrosine phosphatase-alpha overexpression inhibited alpha4beta1-stimulated NB motility and Src activation consistent with alpha4-regulated Src activity occurring through Src Tyr-529 dephosphorylation. In alpha4 shRNA-expressing NB cells, alpha4beta1-stimulated Src activation and NB cell motility were rescued by wild type but not cytoplasmic domain-truncated alpha4 re-expression. These studies, supported by results using reconstituted fibroblasts, reveal that alpha4beta1-mediated Src activation is mechanistically distinct from FAK-mediated Src activation during alpha5beta1-mediated NB migration and support the evaluation of inhibitors to alpha4, Src and FAK in the control of NB tumor progression.

Conserved synteny between the Fugu and human PTEN locus and the evolutionary conservation of vertebrate PTEN function.

Mutations of PTEN, which encodes a protein-tyrosine and lipid phosphatase, are prevalent in a variety of human cancers. The human genome 'draft' sequence still lacks organization and much of the PTEN and adjacent loci remain undefined. The pufferfish, Fugu rubripes, by virtue of having a compact genome represents an excellent template for rapid vertebrate gene discovery. Sequencing of 56 kb from the Fugu pten (fpten) locus identified four complete genes and one partial gene homologous to human genes. Genes neighboring fpten include a PAPS synthase (fpapss2) differentially expressed between non-metastatic/metastatic human carcinoma cell lines, an inositol phosphatase (fminpp1) and an omega class glutathione-S-transferase (fgsto). We have determined the order of human BAC clones at the hPTEN locus and that the locus contains hPAPSS2 and hMINPP1 genes oriented as are their Fugu orthologs. Although the human genes span 500 kb, the Fugu genes lie within only 22 kb due to the compressed intronic and intergenic regions that typify this genome. Interestingly, and providing striking evidence of regulatory element conservation between widely divergent vertebrate species, the compact 2.1 kb fpten promoter is active in human cells. Also, like hPTEN, fpten has a growth and tumor suppressor activity in human glioblastoma cells, demonstrating conservation of protein function.

Interaction of farnesylated PRL-2, a protein-tyrosine phosphatase, with the beta-subunit of geranylgeranyltransferase II.

Protein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the beta-subunit of Rab geranylgeranyltransferase II (betaGGT II). The specific interaction of betaGGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with betaGGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for betaGGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for betaGGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the alpha-subunit (alpha) of GGT II, betaGGT II, and PRL-2 resulted in alpha/betaGGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous alpha/betaGGT II activity in HeLa cells. Together, these results indicate that the binding of alphaGGT II and PRL-2 to betaGGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity.

Calmodulin binds to and inhibits the activity of the membrane distal catalytic domain of receptor protein-tyrosine phosphatase alpha.

cDNA expression library screening revealed binding between the membrane distal catalytic domain (D2) of protein-tyrosine phosphatase alpha (PTPalpha) and calmodulin. Characterization using surface plasmon resonance showed that calmodulin bound to PTPalpha-D2 in a Ca(2+)-dependent manner but did not bind to the membrane proximal catalytic domain (D1) of PTPalpha, to the two tandem catalytic domains (D1D2) of PTPalpha, nor to the closely related D2 domain of PTPepsilon. Calmodulin bound to PTPalpha-D2 with high affinity, exhibiting a K(D) approximately 3 nm. The calmodulin-binding site was localized to amino acids 520-538 in the N-terminal region of D2. Site-directed mutagenesis showed that Lys-521 and Asn-534 were required for optimum calmodulin binding and that restoration of these amino acids to the counterpart PTPepsilon sequence could confer calmodulin binding. The overlap of the binding site with the predicted lip of the catalytic cleft of PTPalpha-D2, in conjunction with the observation that calmodulin acts as a competitive inhibitor of D2-catalyzed dephosphorylation (K(i) approximately 340 nm), suggests that binding of calmodulin physically blocks or distorts the catalytic cleft of PTPalpha-D2 to prevent interaction with substrate. When expressed in cells, full-length PTPalpha and PTPalpha lacking only D1, but not full-length PTPepsilon, bound to calmodulin beads in the presence of Ca(2+). Also, PTPalpha was found in association with calmodulin immunoprecipitated from cell lysates. Thus calmodulin does associate with PTPalpha in vivo but not with PTPalpha-D1D2 in vitro, highlighting a potential conformational difference between these forms of the tandem catalytic domains. The above findings suggest that calmodulin is a possible specific modulator of PTPalpha-D2 and, via D2, of PTPalpha.

Prenylation-dependent association of protein-tyrosine phosphatases PRL-1, -2, and -3 with the plasma membrane and the early endosome.

PRL-1, -2, and -3 represent a novel class of protein-tyrosine phosphatase with a C-terminal prenylation motif. Although PRL-1 has been suggested to be associated with the nucleus, the presence of three highly homologous members and the existence of a prenylation motif call for a more detailed examination of their subcellular localization. In the present study, we first demonstrate that mouse PRL-1, -2, and -3 are indeed prenylated. Examination of N-terminal epitope-tagged PRL-1, -2, and -3 expressed in transiently transfected cells suggests that PRL-1, -2, and -3 are present on the plasma membrane and intracellular punctate structures. Stable Chinese hamster ovary cells expressing PRL-1 and -3 in an inducible manner were established. When cells were treated with brefeldin A, PRL-1 and -3 accumulated in a collapsed compact structure around the microtubule-organizing center. Furthermore, PRL-1 and -3 redistributed into swollen vacuole-like structures when cells were treated with wortmannin. These characteristics of PRL-1 and -3 are typical for endosomal proteins. Electron microscope immunogold labeling reveals that PRL-1 and -3 are indeed associated with the plasma membrane and the early endosomal compartment. Expression of PRL-3 is detected in the epithelial cells of the small intestine, where PRL-3 is present in punctate structures in the cytoplasm. When cells are treated with FTI-277, a selective farnesyltransferase inhibitor, PRL-1, -2, and -3 shifted into the nucleus. Furthermore, a mutant form of PRL-2 lacking the C-terminal prenylation signal is associated with the nucleus. These results establish that the primary association of PRL-1, -2, and -3 with the membrane of the cell surface and the early endosome is dependent on their prenylation and that nuclear localization of these proteins may be triggered by a regulatory event that inhibits their prenylation.

Isolation of a novel protein tyrosine phosphatase inhibitor, 2-methyl-fervenulone, and its precursors from Streptomyces.

High-throughput screening identified an extract from Streptomyces sp. IM 2096 with inhibitory activity toward several protein tyrosine phosphatases (PTPs). Four 1,2,4-triazine compounds 2096A-D (1-4) were isolated from this extract and their structures elucidated by interpretation of spectroscopic data and confirmed by degradation and synthesis. The novel glycocyamidine derivatives 1 and 2 are diastereomers and may interconvert. Both are inactive in the PTP inhibition assay. Compounds 1 and 2 are unstable and partially decompose to 3 and glycocyamidine (5) at room temperature. Compound 3, known as MSD-92 or 2-methyl-fervenulone, is a broad-specificity PTP inhibitor with comparable potency to vanadate. The imidazo[4, 5-e]-1,2,4-triazine (4), inactive in the PTP-inhibition assay, may be a degradation product of 3.

Protein tyrosine phosphatase alpha (PTPalpha) and contactin form a novel neuronal receptor complex linked to the intracellular tyrosine kinase fyn.

Glycosyl phosphatidylinositol (GPI)-linked receptors and receptor protein tyrosine phosphatases (RPTPs), both play key roles in nervous system development, although the molecular mechanisms are largely unknown. Despite lacking a transmembrane domain, GPI receptors can recruit intracellular src family tyrosine kinases to receptor complexes. Few ligands for the extracellular regions of RPTPs are known, relegating most to the status of orphan receptors. We demonstrate that PTPalpha, an RPTP that dephosphorylates and activates src family kinases, forms a novel membrane-spanning complex with the neuronal GPI-anchored receptor contactin. PTPalpha and contactin associate in a lateral (cis) complex mediated through the extracellular region of PTPalpha. This complex is stable to isolation from brain lysates or transfected cells through immunoprecipitation and to antibody-induced coclustering of PTPalpha and contactin within cells. This is the first demonstration of a receptor PTP in a cis configuration with another cell surface receptor, suggesting an additional mode for regulation of a PTP. The transmembrane and catalytic nature of PTPalpha indicate that it likely forms the transducing element of the complex, and we postulate that the role of contactin is to assemble a phosphorylation-competent system at the cell surface, conferring a dynamic signal transduction capability to the recognition element.

Catalytic activation of the membrane distal domain of protein tyrosine phosphatase epsilon, but not CD45, by two point mutations.

Most, if not all, of the catalytic activity of the tandem catalytic domain-containing receptor-like protein tyrosine phosphatases (PTPs) resides in the membrane proximal domains (D1), with little to no activity associated with the membrane distal domains (D2). Two point mutations in the D2 domain of PTPalpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent kinetic properties towards the phosphotyrosine analogue pNPP, and activate PTPalpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As all D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTPepsilon-D2, closely related to PTPalpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant amino acids, exhibits synergistic activation towards pNPP but not towards a phosphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domains with considerable substitutions in D1-invariant amino acids, is not activated by these mutations, even in the context of a third mutation which restores the minimal essential active site sequence C(X(5))R, indicating that additional defects are sufficient to preclude catalysis. The ability of the KNRY and WPD replacements to activate PTPepsilon-D2 and PTPalpha-D2, but not CD45-D2, in conjunction with the extent and nature of their wild-type amino acid substitutions, suggests that these D2 domains are representative of two functionally distinct groups of D2 domain.

Targeted disruption of the tyrosine phosphatase PTPalpha leads to constitutive downregulation of the kinases Src and Fyn.

A role for the receptor-like protein tyrosine phosphatase alpha (PTPalpha) in regulating the kinase activity of Src family members has been proposed because ectopic expression of PTPalpha enhances the dephosphorylation and activation of Src and Fyn [1] [2] [3]. We have generated mice lacking catalytically active PTPalpha to address the question of whether PTPalpha is a physiological activator of Src and Fyn, and to investigate its other potential functions in the context of the whole animal. Mice homozygous for the targeted PTPalpha allele (PTPalpha-/-) and lacking detectable PTPalpha protein exhibited no gross phenotypic defects. The kinase activities of Src and Fyn were significantly reduced in PTPalpha-/- mouse brain and primary embryonic fibroblasts, and this correlated with enhanced phosphorylation of the carboxy-terminal regulatory Tyr527 of Src in PTPalpha-/- mice. Thus, PTPalpha is a physiological positive regulator of the tyrosine kinases Src and Fyn. Increased tyrosine phosphorylation of several unidentified proteins was also apparent in PTPalpha-/- mouse brain lysates. These may be PTPalpha substrates or downstream signaling proteins. Taken together, the results indicate that PTPalpha has a dual function as a positive and negative regulator of tyrosine phosphorylation events, increasing phosphotyrosyl proteins through activation of Src and Fyn, and directly or indirectly removing tyrosine phosphate from other unidentified proteins.

Insulin secretagogues activate the secretory granule receptor-like protein-tyrosine phosphatase IAR.

To investigate the potential role of protein-tyrosine phosphatases (PTPs) in regulated secretion, cellular PTP activity was measured in pancreatic beta cell lines after exposure to insulin secretagogues. A peak of elevated PTP activity was detected in whole cell lysates after 15-20 min of treatment of the cells with high KCl, glucose, or TPA, which did not appear upon treatment with control compounds. Neither was it detected in cells that do not undergo regulated secretion. The PTP activation was transient, SDS-resistant, and localized to the cytoskeleton fraction of cells. The cytoskeletal localization of IAR, a receptor-like PTP associated with secretory granules of neuroendocrine cells, suggested the possibility that IAR is the secretagogue-activated PTP. The transient expression of human IAR in betaTC3 and HIT-T15 beta cells, followed by treatment with secretagogues or control compounds and immunoprecipitation of human IAR, showed that immunoprecipitates from the secretagogue-treated cells contained an elevated PTP activity. The secretagogue-induced activation of IAR had identical kinetics to that of the endogenous PTP. Although ectopic IAR was present in membrane and cytoskeletal fractions from the cells, only the cytoskeleton-associated IAR could be activated. Thus IAR represents the endogenous secretagogue-responsive PTP, or at least a component of it, and is one of the few receptor-like PTPs for which enzymatic activation has been demonstrated. Insulin secretion is detected prior to IAR activation, suggesting that IAR is not required for immediate secretion but likely plays a role in events downstream of insulin secretion or in another pathway related to the specialized function of secretory cells.

Interconversion of the kinetic identities of the tandem catalytic domains of receptor-like protein-tyrosine phosphatase PTPalpha by two point mutations is synergistic and substrate-dependent.

The two tandem homologous catalytic domains of PTPalpha possess different kinetic properties, with the membrane proximal domain (D1) exhibiting much higher activity than the membrane distal (D2) domain. Sequence alignment of PTPalpha-D1 and -D2 with the D1 domains of other receptor-like PTPs, and modeling of the PTPalpha-D1 and -D2 structures, identified two non-conserved amino acids in PTPalpha-D2 that may account for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTPalpha-D1 positively affected the catalytic efficiency of PTPalpha-D2 toward the in vitro substrates para-nitrophenylphosphate and the phosphotyrosyl-peptide RR-src. Together, they synergistically transformed PTPalpha-D2 into a phosphatase with catalytic efficiency for para-nitrophenylphosphate equal to PTPalpha-D1 but not approaching that of PTPalpha-D1 for the more complex substrate RR-src. In vivo, no gain in D2 activity toward p59(fyn) was effected by the double mutation. Alteration of the two corresponding invariant residues in PTPalpha-D1 to those in D2 conferred D2-like kinetics toward all substrates. Thus, these two amino acids are critical for interaction with phosphotyrosine but not sufficient to supply PTPalpha-D2 with a D1-like substrate specificity for elements of the phosphotyrosine microenvironment present in RR-src and p59(fyn). Whether the structural features of D2 can uniquely accommodate a specific phosphoprotein substrate or whether D2 has an alternate function in PTPalpha remains an open question.

Antibodies to the protein tyrosine phosphatases IAR and IA-2 are associated with progression to insulin-dependent diabetes (IDDM) in first-degree relatives at-risk for IDDM.

Insulin-dependent diabetes mellitus (IDDM) is preceded by the presence of antibodies against islet proteins including a protein tyrosine phosphatase (PTP) designated IA-2. Recently, we cloned a novel PTP named IAR which shares 43% sequence identity with IA-2 and is recognised by antibodies from a majority of patients with IDDM. The aim of the present study was to determine whether IAR antibodies (IAR Ab) or IA-2 antibodies (IA-2 Ab) are associated with progression to IDDM in first-degree relatives "at-risk" for IDDM (operationally defined as those with islet cell antibodies [ICA] > or = 20JDFU or insulin autoantibodies [IAA] > or = 100 nU/ml), and to examine combinations of IAR Ab and IA-2 Ab in these subjects. The sensitivity and specificity of these antibodies were also examined in patients with recent-onset IDDM. Using Cox's Proportional Hazards Model, the number of siblings with IDDM was associated with progression to IDDM in "at-risk" relatives, but other covariables (age, sex, number of affected offspring or parents) were not significantly associated. Using number of affected siblings as a covariable, both IAR and IA-2 antibodies were significantly associated with progression to IDDM (p < 0.005). Combinations of both antibodies, however, did not result in a significantly stronger association with progression to IDDM. The threshold of positivity for IAR Ab (0.5 units) and IA-2 Ab (3.0 units) assays was adjusted to give the same specificity (97.9%) for each assay in 144 healthy control subjects, to allow standardised comparisons. Levels of IAR Ab and IA-2 Ab were strongly correlated in 53 recent-onset IDDM patients (r = 0.70, p < 0.0001) but 11.3% had IAR Ab in the absence of IA-2 Ab and 16.9% had IA-2 Ab in the absence of IAR Ab. The sensitivity for IDDM (defined as the proportion of IDDM patients positive) was 56.6% for IAR Ab and 62.3% for IA-2 Ab. We conclude that there is considerable overlap in IA-2 Ab and IAR Ab positivity, although either antibody can occur independently in IDDM patients. Both IAR Ab and IA-2 antibodies are associated with progression to IDDM in first-degree relatives at-risk of IDDM, but the use of IAR and IA-2 antibodies in combination are not significantly more strongly associated with progression than single antibodies. IAR Ab may play an important role in the prediction of IDDM.

Physical and functional interactions between receptor-like protein-tyrosine phosphatase alpha and p59fyn.

We have examined the in vivo activity of receptor-like protein-tyrosine phosphatase alpha (PTPalpha) toward p59(fyn), a widely expressed Src family kinase. In a coexpression system, PTPalpha effected a dose-dependent tyrosine dephosphorylation and activation of p59(fyn), where maximal dephosphorylation correlated with a 5-fold increase in kinase activity. PTPalpha expression resulted in increased accessibility of the p59(fyn) SH2 domain, consistent with a PTPalpha-mediated dephosphorylation of the regulatory C-terminal tyrosine residue of p59(fyn). No p59(fyn) dephosphorylation was observed with an enzymatically inactive mutant form of PTPalpha or with another receptor-like PTP, CD45. Many enzyme-linked receptors are complexed with their substrates, and we examined whether PTPalpha and p59(fyn) underwent association. Reciprocal immunoprecipitations and assays detected p59(fyn) and an appropriate kinase activity in PTPalpha immunoprecipitates and PTPalpha and PTP activity in p59(fyn) immunoprecipitates. No association between CD45 and p59(fyn) was detected in similar experiments. The PTPalpha-mediated activation of p59(fyn) is not prerequisite for association since wild-type and inactive mutant PTPalpha bound equally well to p59(fyn). Endogenous PTPalpha and p59(fyn) were also found in association in mouse brain. Together, these results demonstrate a physical and functional interaction of PTPalpha and p59(fyn) that may be of importance in PTPalpha-initiated signaling events.

Solid-phase synthesis of potential protein tyrosine phosphatase inhibitors via the Ugi four-component condensation.

A library of 108 alpha,alpha-difluoromethylenephosphonic acids was prepared by Ugi four-component condensation using Rink-NH2 resin, 4-[(diethoxyphosphinyl) difluoromethyl]benzoic acid, and a set of 18 aldehydes and 6 isonitriles. Following resin cleavage, the diethylphosphonate esters were hydrolyzed with trimethylsilyl bromide to yield the free acids which were assayed for inhibition of PTP alpha, PTP beta and PTP epsilon.

Kinetic analysis of two closely related receptor-like protein-tyrosine-phosphatases, PTP alpha and PTP epsilon.

Among transmembrane protein-tyrosine-phosphatases, the membrane distal catalytic domain (D2) of protein-tyrosine-phosphatase alpha (PTP alpha) is unusual in having low but detectable activity in the absence of the membrane proximal catalytic domain (D1). To investigate the catalytic properties of PTP alpha D2 in association with D1, kinetic parameters of activity were established for PTP alpha D1D2 proteins containing an inactivating point mutation in D1 and/or D2. In this context, D2 activity was unchanged by the presence (N-terminal or C-terminal) or absence of inactive D1, and the presence or absence of inactive D2 affected the velocity but not the Km of D1 catalysis. While D1 appears to be the major catalytic contributor to PTP alpha activity, D2 possesses a significantly higher substrate-specific activity relative to wild-type D1D2 than the D2 domains of other protein-tyrosine-phosphatases. Also, PTP alpha D2 is an active phosphatase with comparable or better efficiency, on the basis of k(cat)/Km criteria, to some of the dual specificity phosphatases. Kinetic parameters of a closely related receptor-like protein-tyrosine-phosphatase, PTP epsilon, were determined. PTP epsilon D1 is the major, if not the only, catalytic moiety of PTP epsilon, and has much higher turnover numbers than D1 of PTP alpha. The PTP epsilon D2 activity is insignificant compared to that of PTP epsilon-D1D2, with lower turnover numbers than PTP alpha D2. Thus, the intrinsic activity of PTP alpha D2 is high compared to other D2 domains and, more outstandingly, its activity relative to D1 appears unique. These are also apparent upon in vitro assay of full-length PTP alpha catalytic mutants expressed in mammalian cells. Together. these results suggest potential catalytic and regulatory roles for PTP alpha D2, and that PTP alpha may be an optimal model transmembrane protein-tyrosine-phosphatase for investigating the former within the cell.

Cloning and characterization of islet cell antigen-related protein-tyrosine phosphatase (PTP), a novel receptor-like PTP and autoantigen in insulin-dependent diabetes.

Cloning of the cDNA encoding a novel human protein- tyrosine phosphatase (PTP) called islet cell antigen-related PTP (IAR) predicts a receptor-like molecule with an extracellular domain of 614 amino acids containing a hydrophobic signal peptide, one potential N-glycosylation site, and an RGDS peptide which is a possible adhesive recognition sequence. The 376-amino acid intracellular region contains a single catalytic domain. Recombinant IAR polypeptide has phosphatase activity. Northern blot analysis shows tissue-specific expression of two IAR transcripts of 5.5 and 3. 7 kilobases, which are most abundant in brain and pancreas. The IAR PTP is homologous in its intracellular region to IA-2, a putative PTP that is an insulin-dependent diabetes mellitus (IDDM) autoantigen. IAR is also reactive with IDDM patient sera. IAR and IA-2 may distinguish different populations of IDDM autoantibodies since they identify overlapping but nonidentical sets of IDDM patients. Thus IAR is likely to be an islet cell antigen useful in the preclinical screening of individuals for risk of IDDM.

Novel alternative splicing predicts a secreted extracellular isoform of the human receptor-like protein tyrosine phosphatase LAR.

RT-PCR was used to examine the expression of LAR (encoding the leukocyte-common antigen-related protein tyrosine phosphatase) in normal human colon mucosa, and colon polyps and tumors. Although the LAR protein was not detected in the colon in a previous immunohistochemical study, amplification of a region of LAR between the most membrane proximal (eighth) fibronectin type-III (FN-III) repeat and the transmembrane domain demonstrated LAR expression in all samples, but showed no difference in expression within matched samples from each patient examined. An additional minor fragment amplified in all reactions was consistently observed in colon and various cell line samples using this and two other LAR-specific sets of primers. Cloning and sequencing of the fragment identified it as deriving from a novel alternatively spliced form of LAR containing a retained intron of 85 bp. This intron encodes an additional 13 amino acids followed by an in-frame stop codon, thus its retention is predicted to give rise to a secreted LAR extracellular region isoform(s). LAR transcripts containing the intron were detected by RNase protection assay of colon samples and were present in most human tissues examined by Northern analysis. A protein in colon tumor extract was recognized by antiserum raised to the intron-encoded sequence. Soluble isoforms of the LAR extracellular immunoglobulin (Ig)-like/FN-III repeat-containing region could have a biological function distinct from those isoforms localized at the cell surface and/or coupled to intracellular phosphatase activity.

Increased mRNA expression of the receptor-like protein tyrosine phosphatase alpha in late stage colon carcinomas.

The protein tyrosine phosphatase alpha (PTP alpha) mRNA level in paired samples of late stage (Dukes' D) colorectal tumors and adjacent normal colon mucosa was quantified by RNase protection assays. After normalization against 18S RNA or beta-actin mRNA level, a 2-10-fold increase in PTP alpha mRNA was detected in 10 of 14 tumors (approximately 70%) compared to mucosa. In situ hybridization of digoxigenin-labelled antisense PTP alpha RNA to tumor and mucosa sections produced a signal only in neoplastic cells of the tumor sample, consistent with the high increase in PTP alpha mRNA detected by RNase protection assays of some of the tumors. This is the first report suggesting an association of a protein tyrosine phosphatase with colorectal carcinoma. PTP alpha is a receptor-like PTP thought to be involved in regulating cell proliferation. Its oncogenic properties when overexpressed in cultured fibroblasts suggest that PTP alpha overexpression could contribute to the tumorigenic process in colon carcinoma.