Protein kinases and proto-oncogenes: biochemical regulators of the eukaryotic cell cycle

Robert Freeman

Appointment Period: 1987-1991, Grant Years: [02,03,04,05,06]

Robert Freeman Studies from several laboraories have suggested that the c-mos proto-oncogene may have an important function in germ cell development. In 1989, we described a direct role for the Xenopus laevis homolog of c-mos (mos xe) in inducing progressing of meiosis in Xenopus oocytes. During the past year, we have extended these studies. First, we have demonstrated that the v-mos oncogene can function similarly to mos xe by inducing meiotic maturation when expressed in Xenopus oocytes. Second, we addressed the possibility that mos may interact directly with maturation or M-phase promoting factor (MPF), the protein complex responsible for governing entry into M-phase. We demonstrated a dramatic acceleration in GVBD that occurs when both mox xe and cyclin B are over-expressed in the same oocytes. Currently, we are investigating the possibility that the phosphorylation of mos xe during oocyte maturation regultes its protein kinase activity. We have contstructed a series of mutations in mos xe by replacing conserved serine residues with alanines. When assayed for oocyte maturation, one of these mutants was found to be inactive. This mutant is stably expressed and phosphorylated in oocytes, and we are determining whether the point mutation results in the removal of a phosphorylation site.

PUBLICATIONS (resulting from this training, and some recent ones)

Freeman RS, Pickham KM, Kanki JP, Lee BA, Pena SV, Donoghue DJ. (1989) The Xenopus homolog of the mos proto-oncogene transforms mammalian fibroblasts and induces maturation of Xenopus oocytes. Proc. Natl. Acad. Sci. USA 86:5805-5809.

Freeman RS, Donoghue DJ. (1989) Transforming mutant v-mos protein kinases that are deficient in in vitro autophosphorylation. Mol. Cell. Biol. 9:4087-4090.

Freeman RS, Kanki JP, Ballantyne SM, Pickham KM, Donoghue DJ (1990) Effects of the v-mos oncogene on Xenopus development: meiotic induction in oocytes and mitotic arrest in cleaving embryos. J. Cell Biol. 111:533-541.

Freeman RS, Donoghue DJ (1991) Protein kinases and proto-oncogenes: biochemical regulators of the eukaryotic cell cycle. Biochemistry 30:2293-2302.

Freeman RS, Ballantyne SM, Donoghue DJ. (1991) Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mos-xe. Mol Cell Biol. 11:1713-1717.

Freeman RS, Meyer AN, Li J, Donoghue DJ. (1992) Phosphorylation of conserved serine residues does not regulate the ability of mosxe protein kinase to induce oocyte maturation or function as cytostatic factor. J Cell Biol. 116:725-735.

Freeman RS, Estus S, Horigome K, Johnson EM Jr. (1993) Cell death genes in invertebrates and (maybe) vertebrates. Curr Opin Neurobiol. 3:25-31.

Smith CJ, Johnson EM Jr, Osborne P, Freeman RS, Neveu I, Brachet P. (1993) NGF deprivation and neuronal degeneration trigger altered beta-amyloid precursor protein gene expression in the rat superior cervical ganglia in vivo and in vitro. Brain Res Mol Brain Res. 17:328-34.

Freeman RS, Estus S, Johnson EM Jr. (1994) Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of Cyclin D1 during programmed cell death. Neuron. 12:343-55.

Estus S, Zaks WJ, Freeman RS, Gruda M, Bravo R, Johnson EM Jr. (1994) Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis. J Cell Biol. 127:1717-27.

Crowder RJ, Freeman RS. (1998) Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons. J Neurosci. 18:2933-43.

Maggirwar SB, Sarmiere PD, Dewhurst S, Freeman RS. (1998) Nerve growth factor-dependent activation of NF-kappaB contributes to survival of sympathetic neurons. J Neurosci. 18:10356-65.

Crowder RJ, Freeman RS. (1999) The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3-kinase and Akt. J Neurochem. 73:466-75.

Crowder RJ, Freeman RS. (2000) Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal. J Biol Chem. 275:34266-71.

Lipscomb EA, Sarmiere PD, Freeman RS. (2001) SM-20 is a novel mitochondrial protein that causes caspase-dependent cell death in nerve growth factor-dependent neurons. J Biol Chem. 276:5085-92.

Sarmiere PD, Freeman RS. (2001) Analysis of the NF-kappa B and PI 3-kinase/Akt survival pathways in nerve growth factor-dependent neurons. Mol Cell Neurosci. 18:320-31.

Straub JA, Lipscomb EA, Yoshida ES, Freeman RS. (2003) Induction of SM-20 in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death. J Neurochem. 85:318-28.

Freeman RS, Hasbani DM, Lipscomb EA, Straub JA, Xie L. (2003) SM-20, EGL-9, and the EGLN family of hypoxia-inducible factor prolyl hydroxylases. Mol Cells. 16:1-12.

Clifton DR, Rydkina E, Freeman RS, Sahni SK. (2005) NF-kappaB activation during Rickettsia rickettsii infection of endothelial cells involves the activation of catalytic IkappaB kinases IKKalpha and IKKbeta and phosphorylation-proteolysis of the inhibitor protein IkappaBalpha. Infect Immun. 73:155-65.

Freeman RS, Barone MC. (2005) Targeting hypoxia-inducible factor (HIF) as a therapeutic strategy for CNS disorders. Curr Drug Targets CNS Neurol Disord. 4:85-92.

Xie L, Johnson RS, Freeman RS. (2005) Inhibition of NGF deprivation-induced death by low oxygen involves suppression of BIMEL and activation of HIF-1. J Cell Biol. 168:911-20.

Clifton DR, Rydkina E, Huyck H, Pryhuber G, Freeman RS, Silverman DJ, Sahni SK. (2005) Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after Rickettsia rickettsii infection: regulation by nuclear transcription factor NF-kappaB. Int J Med Microbiol. 295:267-78.