Genetic analysis of protein tyrosine phosphatases.

Genetic analysis has enhanced our understanding of the biological roles of many protein tyrosine kinases (PTKs). More recently, studies utilizing both spontaneous mutants and mutants induced by homologous recombination techniques have begun to yield key insights into the role of specific protein tyrosine phosphatases (PTPs) and to suggest how PTKs and PTPs interact. Specific PTPs in Saccharomyces cerevesiae and Schizomyces pombe regulate MAP kinase pathways. Several Drosophila receptor PTPs control axonal targeting pathways, whereas the non-receptor PTP Corkscrew (Csw), plays an essential positive signaling role in multiple developmental pathways directed by receptor PTKs. The vertebrate homolog of Csw, SHP-2, also is required for growth factor signaling and normal development. Finally, very recent studies of other mammalian PTPs suggest that they have critical roles in processes as diverse as hematopoiesis and liver and pituitary development.

Structural determinants of SHP-2 function and specificity in Xenopus mesoderm induction.

SHP-2 is a positive component of many receptor tyrosine kinase signaling pathways. The related protein-tyrosine phosphatase (PTP) SHP-1 usually acts as a negative regulator. The precise domains utilized by SHP-2 to transmit positive signals in vivo and the basis for specificity between SHP-1 and SHP-2 are not clear. In Xenopus, SHP-2 is required for mesoderm induction and completion of gastrulation. We investigated the effects of SHP-2 mutants and SHP-2/SHP-1 chimeras on basic fibroblast growth factor-induced mesoderm induction. Both SH2 domains and the PTP domain are required for normal SHP-2 function in this pathway. The N-terminal SH2 domain is absolutely required, whereas the C-terminal SH2 contributes to wild-type function. The C-terminal tyrosyl phosphorylation sites and proline-rich region are dispensable, arguing against adapter models of SHP-2 function. Although the SH2 domains contribute to SHP-2 specificity, studies of SHP chimeras reveal that substantial specificity resides in the PTP domain. Thus, PTP domains exhibit biologically relevant specificity in vivo, and noncatalytic and catalytic domains of PTPs contribute to specificity in a combinatorial fashion.

Activated mutants of SHP-2 preferentially induce elongation of Xenopus animal caps.

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.

Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.

The application of direct current plasma spectrometry to the study of the fractionation of iron and phosphorus in surface waters.

By use of a direct-current plasma as the excitation source for atomic-emission measurements, samples containing various amounts of particulate matter can be analysed with precisions of better than +/- 10% relative standard deviation provided that particle agglomeration is controlled by acidification and/or filtration. Sample acidification and filtration can have profound effects on the determination of elemental fractionation and distribution in surface waters. By application of a controlled acidification and filtration process, the fraction of an element that is particle-bound and that which is soluble in water can be estimated. The magnitudes of the particle-bound and water-soluble fractions are influenced by a number of factors, including the concentration and average size of the solids in the water, the position of the sample in the water column, and the type of sediment underlying the water column.

The SH2-containing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development.

SH-PTP2, the vertebrate homolog of Drosophila corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. We assayed the effects of injection of wild-type and mutant SH-PTP2 RNAs on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. Our results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase.