Functions of the v-sis and the mos oncogene family and associated genes

Mark Hannink

Appointment Period: 1986-1987, Grant Years: [02]

During my period of support on this Training Grant, I explored the mechanism of cellular transformation exploited by several oncogenes. One of these, the mos protein kinase, represents the transforming gene product encoded by Moloney murine sarcoma virus clone 124. This protein kinase, p37mos, contains a lysine residue (lysine-121) that is conserved among all members of the protein kinase family. This lysine has been shown to be part of a conserved ATP-binding site in both the catalytic subunit of the cAMP-dependent proteinkinase and p60v-src. We created one of the first site-directed mutations in any oncogenic protein kinase in order to determine whether this lysine is required for the transforming activity of p37mos. Two site-specific mutations were constructed, resulting in the substitution of an aspartic acid or arginine codon in place of the codon for lysine-121. Both mutations abolished the ability of the mos gene to transform cells. Our results demonstrated that lysine-121 is required for the ability of p37mos to transform cells and provided evidence that the conserved lysine residue is specifically involved in the catalytic activity of the mos protein kinase, and of protein kinases in general.

I also investigated the v-sis oncogene and its cellular homolog, c-sis, encoding the B-chain of PDGF (platelet-derived growth factor). Cells transformed by v-sis produce a PDGF-related molecule which is able to stimulate the PDGF-Receptor in an autocrine fashion. In one project, I investigated the intracellular trafficking of the v-sis protein, and demonstrated the importance of an unidentified N-terminal signal sequence for correct processing. Subsequently, we examined mutants in the v-sis protein that resulted in either nuclear localization, or targeting to the ER/Golgi. Together with other students in the lab, I was also able to define the minimal region of v-sis required for biological activity, and to demonstrate that N-linked glycosylation was not required for activity.

These studies advanced our understanding of these prototypic oncogenes, helping to understand how both the v-mos and v-sis oncogenes are able to result in abnormal cellular proliferation.

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

Hannink M, Donoghue DJ (1984) Requirement for a signal sequence in biological expression of the v-sis oncogene. Science 226:1197-1199.

Hannink M, Donoghue DJ (1985) Lysine residue #121 in the proposed ATP-binding site of the v-mos protein is required for transformation. Proc. Natl. Acad. Sci. USA, 82:7894-7898.

Hannink M, Donoghue DJ. (1986) Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation and proteolytic processing. Mol. Cell Biol. 6:1343-1348.

Singh B, Hannink M, Donoghue DJ, Arlinghaus RB. (1986) p37mos-associated serine/threonine protein kinase activity correlates with the cellular transformation function of v-mos. J. Virol. 60:1148-1152.

Sauer MK, Hannink M, Donoghue DJ. (1986) Deletions in the N-terminal coding region of the v-sis gene: determination of the minimal transforming region. J. Virol. 59:292-300.

Hannink M, Sauer MK, Donoghue DJ. (1986) Deletions in the C-terminal coding region of the v-sis gene: dimerization is required for transformation. Mol. Cell Biol. 6:1304-1314.

Bold RJ, Hannink M, Donoghue DJ. (1986) Functions of the mos oncogene family and associated gene products. Cancer Surveys 5:243-255.

Hannink M, Donoghue DJ. (1986) Cell surface expression of membrane anchored v-sis gene products: glycosylation is not required for cell surface transport. J. Cell Biol. 103:2311-2322.

Lee BA, Maher DW, Hannink M, Donoghue DJ. (1987) Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules. Mol. Cell. Biol. 7:3527-3537.

Singh BC, Wittenberg C, Hannink M, Reed SI, Donoghue DJ, Arlinghaus RB. (1988) The histidine-221 to tyrosine substitution in v-mos abolishes its biological function and its protein kinase activity. Virology 164:114-120.

Hannink M, Donoghue DJ. (1988) Autocrine stimulation by the v-sis gene product requires a ligand-receptor interaction at the cell surface. J. Cell Biol. 107:287-298.

Hannink M, Donoghue DJ (1989) Structure and function of platelet-derived growth factor (PDGF) and related proteins. BBA Reviews on Cancer 989:1-10.

Hannink M, Temin HM. (1989) Transactivation of gene expression by nuclear and cytoplasmic rel proteins. Mol Cell Biol. 9:4323-36.

Ballard DW, Walker WH, Doerre S, Sista P, Molitor JA, Dixon EP, Peffer NJ, Hannink M, Greene WC. (1990) The v-rel oncogene encodes a kappa B enhancer binding protein that inhibits NF-kappa B function. Cell. 63:803-14.

Walker WH, Stein B, Ganchi PA, Hoffman JA, Kaufman PA, Ballard DW, Hannink M, Greene WC. (1992) The v-rel oncogene: insights into the mechanism of transcriptional activation, repression, and transformation. J Virol. 66:5018-29.

Diehl JA, McKinsey TA, Hannink M. (1993) Differential pp40I kappa B-beta inhibition of DNA binding by rel proteins. Mol Cell Biol. 13:1769-78.

Diehl JA, Hannink M. (1994) Identification of a C/EBP-Rel complex in avian lymphoid cells. Mol Cell Biol. 14:6635-46.

Rottjakob EM, Sachdev S, Leanna CA, McKinsey TA, Hannink M. (1996) PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms. J Virol. 70:3176-88.

Sachdev S, Diehl JA, McKinsey TA, Hans A, Hannink M. (1997) A threshold nuclear level of the v-Rel oncoprotein is required for transformation of avian lymphocytes. Oncogene. 14:2585-94.

Sachdev S, Hannink M. (1998) Loss of IkappaB alpha-mediated control over nuclear import and DNA binding enables oncogenic activation of c-Rel. Mol Cell Biol. 18:5445-56.

Sachdev S, Bagchi S, Zhang DD, Mings AC, Hannink M. (2000) Nuclear import of IkappaBalpha is accomplished by a ran-independent transport pathway. Mol Cell Biol. 20:1571-82.

Lee SH, Hannink M. (2001) The N-terminal nuclear export sequence of IkappaBalpha is required for RanGTP-dependent binding to CRM1. J Biol Chem. 276:23599-606.

Zhang DD, Hannink M. (2003) Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress. Mol Cell Biol. 23:8137-51.

Zhang DD, Lo SC, Cross JV, Templeton DJ, Hannink M. (2004) Keap1 is a redox-regulated substrate adaptor protein for a Cul3- dependent ubiquitin ligase complex. Mol Cell Biol. 24:10941-53.

Zhang DD, Lo SC, Sun Z, Habib GM, Lieberman MW, Hannink M. (2005) Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway. J Biol Chem. 280:30091-9.

Lo SC, Hannink M. (2006) CAND1-mediated substrate adaptor recycling is required for efficient repression of Nrf2 by Keap1. Mol Cell Biol. 26:1235-44.

Lo SC, Li X, Henzl MT, Beamer LJ, Hannink M. (2006) Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling. EMBO J. 25:3605-17.

Lo SC, Hannink M. (2006) PGAM5, a Bcl-XL-interacting protein, is a novel substrate for the redox-regulated Keap1-dependent ubiquitin ligase complex. J Biol Chem. 281:37893-903.