Faculty Bibliography

PTP-1B is a major nontransmembrane phosphotyrosine phosphatase in human cell lines and tissues, but its physiological function(s) and mechanism(s) of regulation are largely unknown. We have found that in human diploid fibroblasts a novel PTP-1B mRNA isoform is produced upon stimulation of quiescent cells with a variety of growth factors. Generation of this isoform requires protein synthesis, suggesting that the product(s) of an immediate early response gene(s) is required. RNase protection and reverse transcription polymerase chain reaction analysis demonstrate that the isoform arises as a consequence of alternative pre-mRNA splicing, leading to retention of the last intron. The novel isoform is found on polyribosomes, indicating that it is actively translated, and is variably expressed in human tissues. Sequence analysis indicates that the isoform encodes a PTP-1B protein with an altered C terminus. To our knowledge, this is the first example of growth factor-regulated alternative splicing.

The non-transmembrane phosphotyrosine phosphatase 1B (PTP-1B) is an abundant enzyme, normally localized to the cytosolic face of the endoplasmic reticulum via a C-terminal targeting sequence. We have found that agonist-induced platelet activation results in proteolytic cleavage of PTP-1B at a site upstream from this targeting sequence, causing subcellular relocation of its catalytic domain from membranes to the cytosol. PTP-1B cleavage is catalyzed by the calcium-dependent neutral protease calpain and is a general feature of platelet agonist-induced aggregation. Moreover, PTP-1B cleavage correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. Engagement of gpIIb-IIIa is necessary for inducing PTP-1B cleavage, suggesting that integrins regulate tyrosine phosphatases as well as tyrosine kinases. PTP-1B cleavage is accompanied by a 2-fold stimulation of its enzymatic activity, as measured by immune complex phosphatase assay, and correlates with discrete changes in the pattern of tyrosyl phosphorylation. Cleavage and subcellular relocation of PTP-1B represents a novel mechanism for altering tyrosyl phosphorylation that may have important physiological implications in cell types other than platelets.

The importance of tyrosyl phosphorylation in regulating growth factor-receptor-mediated signal transduction is firmly established, but the roles of protein tyrosine phosphatases (PTPases) in these pathways are unclear. PTPases that contain src-homology 2 (SH2) domains, which mediate interactions with specific phosphotyrosyl proteins, probably play an important role in early signaling events following growth factor stimulation. In this review, the two mammalian SH2-containing PTPases, SH-PTP1 and SH-PTP2, are described and their possible roles in signal transduction discussed. In addition, the implications of recent genetic studies in the mouse and Drosophila, which shed light on the actions of these PTPases in physiology and pathology, will be addressed.

A human protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity. The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel electrophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis. Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration. Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH optima for peptide substrates were shifted toward neutral. With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower Km (1.7 microM) and 2.4-fold higher kcat (0.11 s-1) than with para-nitrophenyl phosphate. Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor. In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line-weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect.

Much progress has been made in elucidating early events in signal transduction by growth factor receptors with intrinsic tyrosine kinase activity. Upon ligand addition, these receptors dimerize and activate, becoming phosphorylated at a number of tyrosyl residues. These phosphorylation sites serve as docking points for proteins containing src homology-2 (SH2) domains. However, little is known about how phosphotyrosine phosphatases (PTPs), participate in these events. Recently, we and others molecularly cloned a ubiquitously expressed SH2 domain-containing PTP, SH-PTP2 (Syp, PTP1D, PTP2C), and found that it interacts directly with several activated growth factor receptors via its SH2 domains. Using a peptide competition assay, we now demonstrate that the major binding site for SH-PTP2 on the platelet-derived growth factor receptor is phosphotyrosine 1009. Immunoprecipitation studies indicate that SH-PTP2 is the previously unidentified "64-kDa" protein known to bind at this site. Addition of a phosphotyrosyl peptide comprising the region around Tyr-1009 stimulates SH-PTP2 activity 5-10-fold, whereas other phosphotyrosyl peptides from the platelet-derived growth factor receptor have no stimulatory effect. Our data suggest that binding of SH-PTP2 to the activated receptor in vivo should result in stimulation of SH-PTP2 activity.

The pivotal role of tyrosine kinases in signal transduction is well established, but the role of tyrosine phosphatases remains obscure. The discovery of src homology 2 domain-containing protein tyrosine phosphatases suggested roles for these molecules in growth factor signaling pathways, since src homology 2 domains direct association of downstream signaling molecules with activated growth factor receptors and other phosphotyrosyl proteins. We have found that SH-PTP2, a putative homologue of Drosophila corkscrew, associates in vivo with the ligand-activated epidermal growth factor and platelet-derived growth factor receptors. The N-terminal src homology 2 domain of SH-PTP2 directly associates with activated receptors. SH-PTP2 itself is a phosphoprotein, and it becomes tyrosyl phosphorylated upon growth factor activation. These findings suggest several possible models for SH-PTP2 signaling.

We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.

The pGEX glutathione S-transferase (GST) fusion protein system is used extensively for high level expression and rapid purification of fusion proteins from bacterial and eukaryotic cell lysates. Unfortunately, many GST fusion proteins are partially or completely insoluble, and thus cannot be purified efficiently from a crude lysate. We have adapted a protocol, previously used to solubilize actin, for the purification of otherwise insoluble GST fusion proteins. Using a GST fusion of the nontransmembrane protein tyrosine phosphatase 1B, we demonstrate that tyrosine phosphatase enzymatic activity is maintained during the purification process. We provide methods for the purification of GST fusion proteins at analytical and preparative scales, and demonstrate that saturation of glutathione agarose is dependent on fusion protein molecular weight. Finally, we present strategies for eluting purified fusion proteins from glutathione agarose beads, for storing eluted protein, and for preparing covalently coupled affinity matrices.

A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells. By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli. In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E. coli protein. The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE. The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides. The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM. The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides.