Positive feedback between MAP kinase and Mos during Xenopus oocyte maturation.

Mos is a serine-threonine protein kinase and a key regulator of meiosis. One function of Xenopus Mos is to activate mitogen-activated protein kinase (MAPK) through direct phosphorylation and activation of MAPK kinase (MAPKK). All three members of this signal cascade can individually induce hormone-independent reentry of oocytes into meiosis I. However, their inducing efficiency is reduced in the absence of protein synthesis. Here we show that de novo Mos synthesis is required for induction of meiosis I by active MAPKK or Mos-MAPK coinjection. In addition, MAPK efficiently phosphorylates Mos at Ser-3 in vitro. These results suggest that a positive feedback loop exists between MAPK and Mos during oocyte maturation. De novo synthesis of Mos, and other proteins, is required for progression from meiosis I to the metaphase arrest at meiosis II; therefore, one function of MAPK during normal Xenopus oocyte maturation might be to stimulate the synthesis or accumulation of Mos that is required for the completion of meiosis.

Mutagenic analysis of functional domains of the mos proto-oncogene and identification of the sites important for MAPK activation and DNA binding.

We constructed in-frame deletion/replacement mutations in the Xenopus mos proto-oncogene that lie within conserved Mos-specific codons, but outside of the regions that are conserved among the src kinase family of genes. All gene products were assayed in vitro for kinase activity and in vivo for their ability to induce oocyte maturation, embryonic cleavage arrest and cellular transformation. Most mutations in Mos eliminated both kinase and biological activity. However, a mutation in Mos that removed two basic amino acid residues (R94 and K97) downstream from the lysine at the ATP binding site (K90) markedly enhanced autophosphorylation activity. Moreover, this mutant displayed markedly reduced biological activity, lacked transforming activity, and failed to activate mitogen activated protein kinase (MAPK). A second mutant Mos product, lacking amino acids R45-A54, displayed a five-fold increase in cellular transforming activity. This Mos mutant specifically localized to the cytoplasm; in contrast to wild-type (wt) Mos that localized to both the nucleus and the cytoplasm. These data indicate that Mos transforming activity is mediated via signalling exerted in the cytoplasm, presumably through MAPK, and that nuclear localization of the oncogene product interferes with transforming activity. We also show that amino acids R45-A54 are important for Mos DNA binding activity.

Transformation of mammalian cells by constitutively active MAP kinase kinase.

Mitogen-activated protein (MAP) kinase kinase (MAPKK) activates MAP kinase in a signal transduction pathway that mediates cellular responses to growth and differentiation factors. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells by prolonging the activated state of MAPKK and of components downstream in the signaling pathway. To test this hypothesis, constitutively active MAPKK mutants were designed that had basal activities up to 400 times greater than that of the unphosphorylated wild-type kinase. Expression of these mutants in mammalian cells activated AP-1-regulated transcription. The cells formed transformed foci, grew efficiently in soft agar, and were highly tumorigenic in nude mice. These findings indicate that constitutive activation of MAPKK is sufficient to promote cell transformation.

Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes.

Triggering neural cell adhesion molecules of the immunoglobulin superfamily with specific ligands or antibodies inhibited the phosphorylation of tryosyl residues in a subpopulation of alpha- and beta-tubulin associated with membranes from a subcellular fraction of nerve growth cones from fetal rat brain. Preincubation of these membranes with purified extracellular fragments of L1, N-CAM, or myelin-associated glycoprotein, or with antibodies directed against the extracellular domains of L1 or N-CAM, inhibited pp60c-src-dependent phosphorylation of tubulin in an endogenous membrane kinase reaction. Other proteins that affect neurite outgrowth (fibronectin, laminin, antibodies against N-cadherin) had no effect. The results suggest that cell adhesion molecules transduce cell surface events to intracellular signals by modulating the activity of protein tyrosine kinases or phosphatases in axonal membranes to influence cytoskeletal dynamics at the growth cone.

Tubulin is phosphorylated at tyrosine by pp60c-src in nerve growth cone membranes.

We show here that tubulin is the major in vivo substrate of the tyrosine-specific protein kinase pp60c-src in nerve growth cone membranes. Phosphotyrosine antibodies were used to demonstrate phosphotyrosyl residues in a subpopulation of alpha- and beta-tubulin that was highly enriched in a subcellular fraction of growth cone membranes from fetal rat brain. The presence of phosphotyrosine-modified isoforms of alpha- and beta-tubulin in vivo was confirmed by 32p labeling of rat cortical neurons in culture. Tubulin in growth cone membranes was phosphorylated at tyrosine in endogenous membrane phosphorylation reactions (0.068 mol phosphotyrosine/mol alpha-tubulin and 0.045 mol phosphotyrosine/mol beta-tubulin), and phosphorylation was specifically inhibited by antibodies directed against pp60c-src, which is localized in the growth cone membranes. pp60c-src was capable of directly phosphorylating tubulin as shown in immune complex kinase assays with purified brain tubulin. Phosphopeptide mapping revealed a limited number of sites of tyrosine phosphorylation in alpha- and beta-tubulin, with similar phosphopeptides observed in vivo and in vitro. These results reveal a novel posttranslational modification of tubulin that could regulate microtubule dynamics at the growth cone.

Tyrosine phosphorylation of membrane-associated tubulin in nerve growth cones enriched in pp60c-src.

The product of the c-src proto-oncogene (pp60c-src) is a tyrosine-specific protein kinase that is expressed in two phases of neural development. In post-mitotic neuronal cells undergoing terminal differentiation, pp60c-src is present at high levels in the membrane of nerve growth cones and proximal axon shafts. Membrane-associated forms of alpha- and beta-tubulin are the major phosphotyrosine-modified proteins in growth cone membranes in vivo. pp60c-src phosphorylates purified, unassembled tubulin subunits in vitro, inhibiting their ability to polymerize into microtubules. It is conceivable that tubulin phosphorylation by pp60c-src in the growth cone may regulate neurite extension by altering adhesion of cells to the substratum.

Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.

A kinetic analysis of the tyrosine-specific protein kinase of pp60c-src from the C1300 mouse neuroblastoma cell line Neuro-2A and pp60c-src expressed in fibroblasts was carried out to determine the nature of the increased specific activity of the neuroblastoma enzyme. In immune-complex kinase assays with ATP-Mn2+ and the tyrosine-containing peptide angiotensin I as phosphoacceptor substrate, pp60c-src from the neuroblastoma cell line was characterized by a maximum velocity (Vmax.) that was 7-15-fold greater than the Vmax. of pp60c-src from fibroblasts. The neuroblastoma enzyme exhibited Km values for ATP (16 +/- 3 microM) and angiotensin I (6.8 +/- 2.6 mM) that were similar to Km values for ATP (25 +/- 3 microM) and angiotensin I (6.5 +/- 1.7 mM) of pp60c-src from fibroblasts. pp60v-src expressed in Rous-sarcoma-virus-transformed cells exhibited an ATP Km value (25 +/- 4 microM) and an angiotensin I Km value (6.6 +/- 0.5 mM) that approximated the values determined for pp60c-src in neuroblastoma cells and fibroblasts. These results indicate that the pp60c-src kinase from neuroblastoma cells has a higher turnover number than pp60c-src kinase from fibroblasts, and that the neural form of the enzyme would be expected to exhibit increased catalytic activity at the saturating concentrations of ATP that are found intracellularly.