Curtis D. Diltz

Effect of microinjection of a low-Mr human placenta protein tyrosine phosphatase on induction of meiotic cell division in Xenopus oocytes.

Homogeneous preparations of a protein phosphatase that is specific for phosphotyrosyl residues (protein tyrosine phosphatase [PTPase] 1B) were isolated from human placenta and microinjected into Xenopus oocytes. This resulted in an increase in activity of up to 10-fold over control levels, as measured in homogenates with use of an artificial substrate (reduced carboxamidomethylated and maleylated lysozyme). Microinjected PTPase was stable for at least 18 h. It is distributed within the oocyte in a manner similar to the endogenous activity and is suggestive of an interaction with cellular structures or molecules located predominantly in the animal hemisphere. The phosphatase markedly retarded (by up to 5 h) maturation induced by insulin. This, in conjunction with the demonstration that PTPase 1B abolished insulin stimulation of an S6 peptide (RRLSSLRA) kinase concomitant with a decrease in the phosphorylation of tyrosyl residues in a protein with the same apparent Mr as the beta subunit of the insulin and insulinlike growth factor 1 receptors (M. F. Cicirelli, N. K. Tonks, C. D. Diltz, E. H. Fischer, and E. G. Krebs, submitted for publication), provides further support for an essential role of protein tyrosine phosphorylation in insulin action. Furthermore, maturation was significantly retarded even when the PTPase was injected 2 to 4 h after exposure of the cells to insulin. PTPase 1B also retarded maturation induced by progesterone and maturation-promoting factor, which presumably do not act through the insulin receptor. These data point to a second site of action of the PTPase in the pathway of meiotic cell division, downstream of the insulin receptor and following the appearance of active maturation-promoting factor.

Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1

The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling.

Purification of protein-tyrosine phosphatases from human placenta.

Publisher Summary This chapter discusses the purification of protein-tyrosine phosphatases from human placenta. Phosphorylation of proteins on tyrosyl residues is an essential element in the control of normal and neoplastic cell growth. The phosphorylation state of a protein reflects the relative activities of the kinase that phosphorylates it and the phosphatase that removes the phosphate. A major technical difficulty associated with the study of protein phosphatases is the requirement for suitably purified phosphorylated substrates. Characterization of the protein-tyrosine phosphatases (PTPases) provides a necessary complementary perspective for an overall understanding of the control of cellular function by tyrosine phosphorylation. This chapter describes procedures for the preparation of substrates and assay of PTPases. It also discusses the purification of a major low-molecular-weight PTPase from human placenta.

Purification and assay of CD45 : an integral membrane protein-tyrosine phosphatase

Publisher Summary This chapter discusses purification and assay of CD45 of an integral membrane protein-tyrosine phosphatase. The determination of the amino acid sequence of protein-tyrosine phosphatase (PTPase) 1B, a major low-molecular-weight PTPase isolated from human placenta, has demonstrated that this enzyme is not structurally related to the protein-Ser/Thr phosphatases. CD45 represents a family of high-molecular-weight (180k–220k) integral membrane proteins, the expression of which is restricted to cells of the hematopoietic lineage. The molecule can be defined in terms of three segments. The intracellular segment is highly conserved between isoforms and comprises two homologous domains of ∼300 residues, each structurally equivalent to one PTPase molecule. The intracellular and extracellular segments are connected by a single transmembrane hydrophobic stretch of 22 residues. A simple procedure for the purification and assay of CD45 is described in the chapter.