SHP1 and SHP2 protein-tyrosine phosphatases associate with betac after interleukin-3-induced receptor tyrosine phosphorylation. Identification of potential binding sites and substrates.

The cytoplasmic tyrosine phosphatases, SHP1 and SHP2, are implicated in the control of cellular proliferation and survival. Here we demonstrate that both SHP1 and SHP2 associate with the betac subunit of the human interleukin-3 (IL-3) receptor following IL-3 stimulation and that the src homology region 2 (SH2) domains of these phosphatases mediate this interaction. Sequential immunoprecipitation analyses suggest this interaction is direct. Competition studies, using phosphotyrosine-containing peptides based on sequences surrounding key tyrosine residues within betac, suggest that phosphorylation of tyrosine 612 is the key event mediating the association of betac with SHP1 and SHP2. However, inhibition of SHP2 binding to betac, did not prevent tyrosine phosphorylation of SHP2. Interestingly, this same phosphopeptide served as a substrate for the tyrosine phosphatase activity of both SHP1 and SHP2. Binding of these protein-tyrosine phosphatases to the IL-3 receptor may regulate IL-3 signal transduction pathways, both through their catalytic activity and through the recruitment of other molecules to the receptor complex.

Protein-tyrosine phosphatase SH-PTP2 (PTPN11) is localized to 12q24.1-24.3.

A 2.1-kb cDNA probe encoding the human SH2-domain containing protein-tyrosine phosphatase SH-PTP2 (PTPN11) was hybridized to human metaphase chromosomes in three independent experiments. In each instance, hybridization was maximal to chromosome 12q24.1-q24.3. The presence of SH-PTP2 cDNA crosshybridizing sequences located on a number of other chromosomes suggested that SH-PTP2-related genes or pseudogenes are present in the human genome.

Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates.

SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.

A protein kinase isolated from porcine brain microvessels is similar to a class of heat-shock proteins.

To further characterize a protein kinase present in porcine brain microvessels, a cDNA library using porcine microvessel poly(A) RNA was screened with polyclonal antibodies raised against the native protein kinase. Since no full-length cDNA clone could be obtained, the missing sequence information was completed using two subsequent polymerase chain reactions. The amplified transcripts were cloned and the sequence determined. Additionally, a genomic DNA library from porcine kidney was screened to substantiate the results obtained from the polymerase chain reaction. Earlier hints of a relation to a subclass of the family of heat-shock proteins (HSPs) based upon a close sequence similarity at its amino-terminus could be confirmed by comparison of the full-length cDNA sequences. Common protein kinase consensus sequences, a targeting sequence for proteins of the endoplasmic reticulum at the carboxy-terminus as well as a hydrophobic leader sequence in the amino-terminal region of the protein could also be identified. Furthermore, a set of membrane-associated substrate proteins of this enzyme could be detected in brain capillaries. The results indicate that at least some members of the HSP 90 subfamily undergo autophosphorylation and show protein kinase activity by phosphorylating substrate proteins in vitro.

Interleukin (IL)-3 and granulocyte/macrophage colony-stimulating factor, but not IL-4, induce tyrosine phosphorylation, activation, and association of SHPTP2 with Grb2 and phosphatidylinositol 3'-kinase.

Binding of interleukin (IL)-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) to their high affinity cell surface receptors induces tyrosine phosphorylation of a similar set of protein substrates. We have identified one of these common substrates (p70) as the protein-tyrosine phosphatase SHPTP2. The Src homology 2 (SH2) domain of the adaptor protein Grb2 bound with high affinity to tyrosine-phosphorylated SHPTP2 following treatment of cells with IL-3 or GM-CSF, but not IL-4. This interaction was inhibited by two phosphotyrosine peptides, based on sequences within SHPTP2, which conform to the postulated consensus sequence for Grb2 SH2 recognition. Following treatment with IL-3 or GM-CSF, but not IL-4, SHPTP2 co-immunoprecipitated with antibodies directed against the p85 subunit of PI 3'-kinase. This was partially blocked by the same phosphopeptides that blocked Grb2-SH2 binding to SHPTP2. Importantly, treatment with IL-3 resulted in a 2-3-fold increase in SHPTP2 phosphatase activity. These results suggest that SHPTP2 may play an important role in integrating signals from the IL-3 and GM-CSF receptors to both Ras and PI 3'-kinase.

Suicide inactivation of human prostatic acid phosphatase and a phosphotyrosine phosphatase.

4-Difluoromethylphenyl bis(cyclohexylammonium) phosphate was synthesized in 4 steps starting from dibenzyl phosphite and shown to be a time-dependent suicide inactivator of human prostatic acid phosphatase and the SHP protein tyrosine phosphatase. The inactivation of human prostatic acid phosphatase followed pseudo-first-order kinetics with inactivation constants of Ki = 1.0 mM; ki = 0.15 min-1 (t1/2 = 4.6 min at saturation). Phenyl phosphate protected the enzyme against inactivation, indicating that inactivation occurs in the active site. The inactivation of SHP also followed pseudo-first-order kinetics, with a t1/2 = approximately 15 min in the presence of 8.2 mM inhibitor. The mechanism of inactivation likely involves the enzymatic release of difluoromethyl phenol which rapidly eliminates fluoride, generating a quinone methide. This potent electrophile then reacts with residues at the active site of the enzyme. This inhibitor and peptidic derivatives thereof have excellent potential for selective inactivation and labeling of protein tyrosine phosphatases.

Characterization of protein tyrosine phosphatase SH-PTP2. Study of phosphopeptide substrates and possible regulatory role of SH2 domains.

The src homology 2 (SH2) domain containing protein-tyrosine-phosphatase SH-PTP2, was over-expressed in Escherichia coli for a kinetic study employing a set of synthetic 13- to 14-mer phosphopeptide substrates. The full-length SH-PTP2 protein, as well as a truncated form, lacking the two amino terminus SH2 domains (SH-PTP2(delta SH2)), exhibited Michaelis-Menten kinetics, and demonstrated striking substrate preferences on phosphopeptides having sequences based on sites of intracellular protein tyrosine phosphorylation. For example, while a KM of 59 microM and kcat/KM of 1.1 x 10(5) were obtained using SH-PTP2(delta SH2) and PDGFRY1021, a phosphorylation site within the platelet-derived growth factor receptor, other peptides revealed no detectable phosphate release. PDGFRY1009, modeled after a sequence identified as an in vivo binding site for SH-PTP2, was also a good substrate for this enzyme. The truncated form, lacking the SH2 domains demonstrated higher catalytic efficiency than the full-length enzyme. Interestingly, soluble SH2 domains were found to inhibit the catalytic activity of SH-PTP2 in a concentration-dependent manner. There was also evidence of a non-phosphotyrosine-mediated association between the two domains. These observations suggested that the SH2 domains have a direct role in regulating the catalytic activity of SH-PTP2.

Isolation and partial characterization of an 80,000-dalton protein kinase from the microvessels of the porcine brain.

A novel serine/threonine-specific protein kinase was isolated from the microvessels of porcine brains. The molecular mass of the protein is 80,000 daltons, as judged by gel electrophoresis under denaturing conditions, or 122,000 daltons, on high-resolution gel permeation chromatography in the native state. The activity of this enzyme is stimulated by various histones or polyamines, like spermine or spermidine, but not by any of the common second messengers. The amino-terminal sequence data show no homologies to any of the published kinases, but rather to a heat-shock protein of unknown function.