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A p38MAPKhomolog Mipk (meiosis-inhibited protein kinase) was cloned from seastar oocytes. This 40-kDa protein shares approximately 65% amino acid identity with mammalian p38-α isoforms. Mipk was one of the major tyrosine-phosphorylated proteins in immature oocytes arrested at the G2/M transition of meiosis I. The tyrosine phosphorylation of Mipk was increased in response to anisomycin, heat, and osmotic shock of oocytes. During 1-methyladenine-induced oocyte maturation, Mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately 12 h after fertilization. At the time of differentiation and acquisition of G phases in the developing embryos, Mipk was rephosphorylated on tyrosine. In oocytes that were microinjected with Mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized, differentiation was blocked. Because MipK antisense oligonucleotides and a dominant-negative (K62R)Mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown, inhibition of Mipk function was not sufficient by itself to cause oocyte maturation. These findings point to a putative role for Mipk in cell cycle control as a G-phase-promoting factor.

Chlorpromazine (25 μM) and trifluoperazine (25 μM) inhibited by 5-fold the activity of CTP: phosphocholine cytidylyltransferase, the rate-limiting enzyme for phosphatidylcholine biosynthesis, in rat liver cytosol. Addition of saturating amounts of rat liver phospholipid to the enzyme assay rapidly reversed the drug-mediated inhibition. Three-fold or greater concentrations of these drugs were required to produce a 50% inhibition of the microsomal cytidylyltransferase. Incubation of rat hepatocytes with 20 μM trifluoperazine or chlorpromazine did not inhibit phosphatidylcholine biosynthesis. These results provide additional evidence for the hypothesis that the active form of cytidylyltransferase is on the endoplasmic reticulum and the enzyme in cytosol appears to be latent.

A synthetic peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, the structure of which is based on that of a phosphorylated sequence in ribosomal protein S6, was employed as a probe for stimulated kinase activity in Xenopus laevisoocytes induced to mature with insulin or progesterone. Insulin elicited an early (20–30 min) 3-fold stimulation of S6 peptide phosphorylating activity that was not evident with progesterone. However, both hormones produced a delayed 7–12-fold stimulation of S6 peptide phosphorylating activity at the time of germinal vesicle breakdown. The results of DEAE-Sephacel, Sephacryl S-200, TSK-400, and heparin-Sepharose chromatographic fractionation experiments imply that a common S6 peptide kinase is activated as a consequence of short and long term insulin exposure, as well as in long term progesterone treatment of oocytes. Omission of potassium from the oocyte culture medium greatly facilitated insulin-induced meiotic maturation.

Allogeneic bone marrow transplant (BMT) recipients have increased susceptibility to infections for prolonged periods after phenotypic reconstitution of donor cells. This immunodeficiency status is characterized by multiple T-cell functional abnormalities. This study was designed to investigate several signaling pathways involved in T-cell activation during this period of immune deficiency. In initial experiments using equal numbers of CD3+cells or highly purified T-cell subpopulations obtained from normal controls and BMT recipients, we confirmed that abnormal T-cell proliferation after CD3 cross-linking, phytohemagglutinin stimulation, or phorbol myristate acetate (PMA) stimulation of peripheral blood mononuclear cells from BMT recipients was due to a qualitative T-cell deficiency rather than to low numbers of circulating T cells. We next investigated the ability of the T-cell receptor/CD3 complex to transduce signals via receptor-associated protein tyrosine kinases. In all BMT recipients, CD3 cross-linking induced protein tyrosine phosphorylation of several proteins in a similar fashion to that seen in controls, including phosphorylation of a 21-kD protein that represents the ζ subunit of the receptor itself. Further investigation showed that CD3 cross-linking and PMA stimulation did not increase 42-44-kD mitogen-activated protein kinase (MAPK) activity. The failure of MAPK activation in BMT recipients occurred despite tyrosine phosphorylation of the 42-44-kD proteins, which, in normal controls, parallels enzyme activation. Our results indicate that T-cell immunodeficiency in BMT recipients is associated with a selective failure of MAPK activation, possibly related to abnormal posttranslational positive regulation of this enzyme.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), lnterleukin-3 (IL-3), and Steel Factor (SF) induce proliferation of hematopoietic cells through binding to specific, high-affinity, cell surface receptors. However, little is known about postreceptor signal transduction pathways. In previous studies, we noted that each of these three factors could independently support proliferation of the human M07 cell line, and also that each factor induced a rapid increase in protein-tyrosyl phosphorylation. Although the proteins phos-phorylated on tyrosine by GM-CSF and IL-3 are similar or identical in MO7 cells, many of the proteins that are phosphorylated on tyrosine after SF are different. However, two proteins, p42 and p44, were prominently phosphorylated in response to all three of the factors. In M07 cells, the tyrosyl phosphorylation of p42 and p44 was transient, peaking at 5 to 15 minutes. In contrast to many of the other proteins which are tyrosyl phosphorylated in response to these factors, phosphorylation of p42 and p44 was temperature-dependent, occurring at 37°C, but not at 4°C. We identified the p42 protein as p42 Mitogen-Activated Protein Kinase (p42maPk, ERK-2) and the p44 as a p42mapk-related protein using monospecific antisera to MAP kinase. GM-CSF, IL-3, and SF were each found to induce MAP kinase activity when assayed in vitro using myelin basic protein (MBP) as a substrate. Remarkably, we found that GM-CSF-induced tyrosyl phosphorylation of p42 and p44 even in nonprolifera-tive cells (neutrophils) that respond to this CSF, and that p42 and p44 were two of the most prominently tyrosyl phosphorylated proteins following GM-CSF stimulation of these cells. These results implicate p42maPkand p44 as important signal transducing molecules in myeloid cells, and it is likely that these kinases play a role as part of a sequential “kinase cascade” linking growth factor receptors to mitogenesis and other cellular responses.

Interleukin-12 (IL-12) is a novel cytokine that enhances numerous functional activities of human T cells and natural killer (NK) cells. The present studies were undertaken to characterize some of the early signaling events following IL-12 stimulation of mitogen-activated normal T cells. In these cells, IL-12 induces rapid tyrosine phosphorylation of proteins of 21, 44, and 54 kD. However, IL-12 does not induce tyrosine phosphorylation in normal resting T cells. In conjunction with increased tyrosine phosphorylation of several substrates, IL-12 stimulation resulted in increased in vitro kinase activity of immunoprecipitated tyrosine phosphorylated proteins. The 44-kD protein has been characterized as one isoform of the mitogen-activated protein (MAP) kinase family. Increased tyrosine phosphorylation of MAP kinase following IL-12 stimulation was also associated with enhanced enzymatic activity of this protein in vitro as measured by myelin basic protein phosphotransferase assay. These studies identify MAP kinase as one of the intracellular elements of the IL-12 signaling pathway in human T cells.