Faculty Bibliography

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.

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.

We have used panels of somatic cell hybrids and fluorescent in situ hybridization to determine the chromosomal localization of the novel nontransmembrane tyrosine phosphatase PTPN6 (protein tyrosine phosphatase, nonreceptor type 6), which contains two SH2 domains. PTPN6 maps to 12p13, a region commonly involved in leukemia-associated chromosomal abnormalities. Since PTPN6 is expressed at high levels in hematopoietic cells of all lineages and its expression is induced early in hematopoietic differentiation, altered expression and/or structure of PTPN6 may play a role in leukemogenesis.

We report the first intracellular characterization of an endogenous nontransmembrane protein tyrosine phosphatase (PTP). Using affinity-purified polyclonal antibodies, we have identified PTP-1B as a 50 kd serine phosphoprotein in immunoprecipitation and immunoblotting assays. Surprisingly, indirect immunofluorescence experiments indicate that PTP-1B is localized predominantly in the endoplasmic reticulum (ER). Subcellular fractionation is consistent with this localization and establishes that PTP-1B is tightly associated with microsomal membranes, with its phosphatase domain oriented towards the cytoplasm. The C-terminal 35 amino acids of PTP-1B are both necessary and sufficient for targeting to the ER. The finding of a tyrosine phosphatase on the ER suggests new possibilities for cellular events controlled by tyrosine phosphorylation.