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

Mark Kamps

Appointment Period: 1985-1987, Grant Years: [01,02]

Mark KampsThroughout the time that Mark Kamps was supported by this training grant, his research focused on understanding the role of tyrosine protein phosphorylation in the proliferation of cells. Eight distinct oncogenes carried by sarcoma viruses encode tyrosine protein kinases. The expression of these enzymes in fibroblasts induces continuous cell division that is independent of the density of those cells and causes cells to grow in soft agar. These are two of the properties most closely associated with that of the transformed phenotype. A second family of tyrosine protein kinases are represented by normal cellular receptors for growth factors. The mitogenic peptides, epidermal growth factor, platelet-derived growth factor, insulin, and insulin-like growth factor I bind to cellular receptors that are tyrosine protein kinases and insodoing stimulate the phosphotransferase activity of these enzymes. In order to understand the mechanism by which tyrosine protein phosphorylation stimulates cell division requires that the substrates of these kinases be isolated and their activities characterized. To approach this task, antibodies were prepared that specifically recognize the hapten, phosphotyrosine. These antibodies stain 10-50 proteins on immunoblots of total cellular proteins from cells transformed by the oncogenes src, yes, fps, abl, fgr, but stain only 3 proteins from normal cells. Approximately eight of these proteins are phosphorylated in cells transformed by each of these oncogenes. These phosphoproteins are being isolated by affinity chromotography with monoclonal antibodies to phosphotyrosine, and once isolated antibodies will be raised to each, and their properties in normal and transformed cells will be characterized. By this approach it is hoped that a small number of proteins can be identified whose phosphorylation appears essential for transformation and which may also be phosphorylated by growth factor receptors upon binding their conjugate ligand.

To pursue the idea that altered tyrosine protein phosphorylation may be involved in the establishment or progression of human cancers, antibodies against phosphotyrosine have been used to identify proteins that are phosphorylated on tyrosine in human malignancies. These antibodies stained proteins of 125 and 145 kDa on immunoblots of total cellular lysates from five of eight squamous cell carcinoma cell lines. In three of six cell lines derived from adenocarcinomas of the colon and in one additional squamous cell carcinoma, a major band at 125 kDa was also stained by the antibody. Neither p125 nor p145 was phosphorylated in normal breast epithelial cells or in colonic mucosal cells. Since preliminary experiments indicate that the phosphorylation of these two proteins is a frequently observed transformation-specific event in these human carcinomas, these proteins will be isolated and characterized as to whether they are phosphorylated in primary tumors as well. Antibodies to phosphotyrosine represent an ideal reagent to examine tyrosine-specific phosphorylation in primary human tumors because the immunoblot analysis simply requires cellular proteins denatured in SDS.

We have also established a number of collaborations. The first involves the use of antiphosphotyrosine antibodies to analyze the substrates of the insulin receptor in an effort to understand the cellular events induced by this hormone. The second investigates the substrates of the neu oncogene, which encodes a receptor-like tyrosine protein kinase and is activated by mutation in a majority of neurobloastomas induced by the chemical mutagen ethylnitosourea.

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

Kamps MP, Buss JE, Sefton BM. (1985) Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc Natl Acad Sci U S A. 82:4625-8.

Kamps MP, Sefton BM. (1986) Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src. Mol Cell Biol. 6:751-7.

Kamps MP, Buss JE, Sefton BM. (1986) Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. Cell. 45:105-12.

Buss JE, Kamps MP, Gould K, Sefton BM. (1986) The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity. J Virol. 58:468-74.

Kamps MP, Sefton BM. (1988) Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 2:305-15.

Kamps MP, Sefton BM. (1989) Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal Biochem. 176:22-7.

Kamps MP, Taylor SS, Sefton BM. (1984) Direct evidence that oncogenic tyrosine kinases and cyclic AMP-dependent protein kinase have homologous ATP-binding sites. Nature. 310:589-92.

Buss JE, Kamps MP, Sefton BM. (1984) Myristic acid is attached to the transforming protein of Rous sarcoma virus during or immediately after synthesis and is present in both soluble and membrane-bound forms of the protein. Mol Cell Biol. 4:2697-704.

Kamps MP, Buss JE, Sefton BM. (1985) Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc Natl Acad Sci U S A. 82:4625-8.

Kamps MP, Sefton BM. (1986) Neither arginine nor histidine can carry out the function of lysine-295 in the ATP-binding site of p60src. Mol Cell Biol. 6:751-7.

Kamps MP, Buss JE, Sefton BM. (1986) Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. Cell. 45:105-12.

Buss JE, Kamps MP, Gould K, Sefton BM. (1986) The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity. J Virol. 58:468-74.

Kamps MP, Sefton BM. (1988) Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 2:305-15.

Stern DF, Kamps MP. (1988) EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions. EMBO J. 7:995-1001.

Glenney JR Jr, Zokas L, Kamps MP. (1988) Monoclonal antibodies to phosphotyrosine. J Immunol Methods. 109:277-85.

Kamps MP, Sefton BM. (1988) Most of the substrates of oncogenic viral tyrosine protein kinases can be phosphorylated by cellular tyrosine protein kinases in normal cells. Oncogene Res. 3:105-15.

Kamps MP, Sefton BM. (1989) Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal Biochem. 176:22-7.

Kamps MP, Murre C, Sun XH, Baltimore D. (1990) A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL. Cell. 60:547-55.

Kamps MP. (1991) Generation and use of anti-phosphotyrosine antibodies for immunoblotting. Methods Enzymol. 201:101-10.

Kamps MP, Look AT, Baltimore D. (1991) The human t(1;19) translocation in pre-B ALL produces multiple nuclear E2A-Pbx1 fusion proteins with differing transforming potentials. Genes Dev. 5:358-68.

Privitera E, Kamps MP, Hayashi Y, Inaba T, Shapiro LH, Raimondi SC, Behm F, Hendershot L, Carroll AJ, Baltimore D, et al. (1992) Different molecular consequences of the 1;19 chromosomal translocation in childhood B-cell precursor acute lymphoblastic leukemia. Blood. 79:1781-8.

Kamps MP, Baltimore D. (1993) E2A-Pbx1, the t(1;19) translocation protein of human pre-B-cell acute lymphocytic leukemia, causes acute myeloid leukemia in mice. Mol Cell Biol. 13:351-7.

Wright DD, Sefton BM, Kamps MP. (1994) Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia. Mol Cell Biol. 14:2429-37.

Kamps MP, Wright DD. (1994) Oncoprotein E2A-Pbx1 immortalizes a myeloid progenitor in primary marrow cultures without abrogating its factor-dependence. Oncogene. 9:3159-66.

Lu Q, Knoepfler PS, Scheele J, Wright DD, Kamps MP. (1995) Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes. Mol Cell Biol. 15:3786-95.

Ogo A, Waterman MR, Kamps MP, Kagawa N. (1995) Protein kinase A-dependent transactivation by the E2A-Pbx1 fusion protein. J Biol Chem. 270:25340-3.

Lu Q, Kamps MP. (1996) Selective repression of transcriptional activators by Pbx1 does not require the homeodomain. Proc Natl Acad Sci U S A. 93:470-4.

Lu Q, Kamps MP. (1996) Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex. Mol Cell Biol. 16:1632-40.

Knoepfler PS, Lu Q, Kamps MP. (1996) Pbx-1 Hox heterodimers bind DNA on inseparable half-sites that permit intrinsic DNA binding specificity of the Hox partner at nucleotides 3' to a TAAT motif. Nucleic Acids Res. 24:2288-94.

Lu Q, Kamps MP. (1997) Heterodimerization of Hox proteins with Pbx1 and oncoprotein E2a-Pbx1 generates unique DNA-binding specifities at nucleotides predicted to contact the N-terminal arm of the Hox homeodomain--demonstration of Hox-dependent targeting of E2a-Pbx1 in vivo. Oncogene. 14:75-83.

Knoepfler PS, Kamps MP. (1997) The highest affinity DNA element bound by Pbx complexes in t(1;19) leukemic cells fails to mediate cooperative DNA-binding or cooperative transactivation by E2a-Pbx1 and class I Hox proteins - evidence for selective targetting of E2a-Pbx1 to a subset of Pbx-recognition elements. Oncogene. 14:2521-31.

Knoepfler PS, Calvo KR, Chen H, Antonarakis SE, Kamps MP. (1997) Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1. Proc Natl Acad Sci U S A. 94:14553-8.

Fu X, McGrath S, Pasillas M, Nakazawa S, Kamps MP. (1999) EB-1, a tyrosine kinase signal transduction gene, is transcriptionally activated in the t(1;19) subset of pre-B ALL, which express oncoprotein E2a-Pbx1. Oncogene. 18:4920-9.

Calvo KR, Knoepfler P, McGrath S, Kamps MP. (1999) An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1. Oncogene. 18:8033-43.

Calvo KR, Sykes DB, Pasillas M, Kamps MP. (2000) Hoxa9 immortalizes a granulocyte-macrophage colony-stimulating factor-dependent promyelocyte capable of biphenotypic differentiation to neutrophils or macrophages, independent of enforced meis expression. Mol Cell Biol. 20:3274-85.

Knoepfler PS, Sykes DB, Pasillas M, Kamps MP. (2001) HoxB8 requires its Pbx-interaction motif to block differentiation of primary myeloid progenitors and of most cell line models of myeloid differentiation. Oncogene. 20:5440-8.

Calvo KR, Knoepfler PS, Sykes DB, Pasillas MP, Kamps MP. (2001) Meis1a suppresses differentiation by G-CSF and promotes proliferation by SCF: potential mechanisms of cooperativity with Hoxa9 in myeloid leukemia. Proc Natl Acad Sci U S A. 98:13120-5.

Klappacher GW, Lunyak VV, Sykes DB, Sawka-Verhelle D, Sage J, Brard G, Ngo SD, Gangadharan D, Jacks T, Kamps MP, Rose DW, Rosenfeld MG, Glass CK. (2002) An induced Ets repressor complex regulates growth arrest during terminal macrophage differentiation. Cell. 109:169-80.

Calvo KR, Sykes DB, Pasillas MP, Kamps MP. (2002) Nup98-HoxA9 immortalizes myeloid progenitors, enforces expression of Hoxa9, Hoxa7 and Meis1, and alters cytokine-specific responses in a manner similar to that induced by retroviral co-expression of Hoxa9 and Meis1. Oncogene. 21:4247-56.

Sykes DB, Scheele J, Pasillas M, Kamps MP. (2003) Transcriptional profiling during the early differentiation of granulocyte and monocyte progenitors controlled by conditional versions of the E2a-Pbx1 oncoprotein. Leuk Lymphoma. 44:1187-99.

Wang GG, Pasillas MP, Kamps MP. (2005) Meis1 programs transcription of FLT3 and cancer stem cell character, using a mechanism that requires interaction with Pbx and a novel function of the Meis1 C-terminus. Blood. 106:254-64.

Hacker H, Redecke V, Blagoev B, Kratchmarova I, Hsu LC, Wang GG, Kamps MP, Raz E, Wagner H, Hacker G, Mann M, Karin M. (2006) Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6. Nature. 439:204-7. Epub 2005 Nov 23.

Wang GG, Pasillas MP, Kamps MP. (2007) Persistent transactivation by meis1 replaces hox function in myeloid leukemogenesis models: evidence for co-occupancy of meis1-pbx and hox-pbx complexes on promoters of leukemia-associated genes. Mol Cell Biol. 26:3902-16.

Lichtenauer UD, Duchniewicz M, Kolanczyk M, Hoeflich A, Hahner S, Else T, Bicknell AB, Zemojtel T, Stallings NR, Schulte DM, Kamps MP, Hammer GD, Scheele JS, Beuschlein F. (2006) Pre-B-cell transcription factor 1 and steroidogenic factor 1 synergistically regulate adrenocortical growth and steroidogenesis. Endocrinology. 148:693-704.