Faculty Bibliography

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.

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.

src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction.

Early-growth-response genes, also known as immediate-early genes, play important roles in regulating cell proliferation. We have identified a new type of early-growth-response gene product, a 77,811-Da putative serine/threonine kinase, which is highly inducible by serum and phorbol ester. mRNA encoding this putative kinase is markedly elevated within 1 h after treatment with mitogen, and this induction is synergistically increased by cycloheximide. Dexamethasone blocks serum induction of the kinase mRNA, as does transformation by v-Ki-ras. The kinase mRNA was detected in mouse brain, lung, and heart. This new putative kinase, which we term Snk, for serum-inducible kinase, showed similarity in its proposed catalytic domain to many other protein kinases; however, no other kinase showed enough sequence similarity with Snk to suggest the existence of a common function. Hence, Snk represents a new type of protein kinase involved in the early mitogenic response whose activity is transcriptionally and posttranscriptionally regulated.

The stimulation of a variety of cell surface receptors promotes the accumulation of the active, GTP-bound form of Ras proteins in cells. This is a critical step in signal transduction because inhibition of Ras activation by anti-Ras antibodies or dominant inhibitory Ras mutants blocks many of the effects of these receptors on cellular function. To reach the active GTP-bound state, Ras proteins must first release bound GDP. This rate-limiting step in GTP binding is thought to be catalysed by a guanine-nucleotide-releasing factor (GRF). Here we report the cloning of complementary DNAs from a rat brain library that encode a approximately 140K GRF for Ras p21 (p140Ras-GRF). Its carboxy-terminal region is similar to that of CDC25, a GRF for Saccharomyces cerevisiae RAS. This portion of Ras-GRF accelerated the release of GDP from RasH and RasN p21 in vitro, but not from the related RalA, or CDC42Hs GTP-binding proteins. A region in the amino-terminal end of Ras-GRF is similar to both the human breakpoint cluster protein, Bcr, and the dbl oncogene product, a guanine-nucleotide-releasing factor for CDC42Hs. An understanding of Ras-GRF function will enhance our knowledge of the many signal transduction pathways mediated by Ras proteins.