[CANCER RESEARCH 63, 8132– 8137, December 1, 2003] Advances in Brief Functional Analysis of Mutations within the Kinase Activation Segment of B-Raf in Human Colorectal Tumors Tsuneo Ikenoue, 1,2 Yohko Hikiba, 1 Fumihiko Kanai, 2 Yasuo Tanaka, 2 Jun Imamura, 2 Takaaki Imamura, 2 Miki Ohta, 2 Hideaki Ijichi, 2 Keisuke Tateishi, 2 Takayuki Kawakami, 2 Jun Aragaki, 1 Masayuki Matsumura, 1,2 Takao Kawabe, 2 and Masao Omata 2 1 Division of Gastroenterology, The Institute for Adult Diseases, Asahi Life Foundation, and 2 Department of Gastroenterology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Abstract Mutations in the B-Raf gene have been reported in a number of human cancers, including colorectal carcinoma. More than 80% of the B-Raf mutations were V599E. Although other mutations have been reported, their functional consequences were unclear. Here, we examined the effect of colon tumor-associated B-Raf mutations within the kinase activation segment, including V599E, on extracellular signal-regulated kinase (Erk) and nuclear factor B (NFB) signaling, and on the transformation of NIH3T3 fibroblasts. Among the six mutations examined, only the B-Raf V599E and K600E mutations greatly increased Erk and NFB signaling, and the transformation of NIH3T3 cells. The B-Raf F594L mutation moderately elevated Erk signaling and NIH3T3 transformation, but did not significantly increase NFB signaling. Although the basal kinase activity of the B-Raf T598I mutant was comparable with that of wild-type, its oncogenic Ras-induced kinase activity was decreased to 60% of wild- type activity. The B-Raf D593V and G595R mutants showed severely reduced kinase activity and affected neither NFB signaling nor NIH3T3 transforming activity. These results suggest that the B-Raf activation segment mutations other than V599E reported in colorectal tumors do not necessarily contribute to carcinogenesis by increasing kinase and trans- forming activities. Introduction The Raf serine/threonine-specific kinases serve as a key signal transducer in the Erk 3 cascade, which regulates diverse physiological processes including cell growth, differentiation, and apoptosis (1– 4). Activation of the small GTPase protein Ras recruits Raf to the plasma membrane, where it becomes activated. Activated Raf proteins di- rectly phosphorylate and activate the downstream kinase MEK, which, in turn, phosphorylates Erk and thereby activates Erk. Acti- vated Erk phosphorylates many cytoplasmic target proteins, such as Rsk, and several nuclear transcription factors, including Elk-1. Raf-1, one of the Raf family of proteins, has been shown to activate NFB transcription factor. NFB plays an important role in distinct cellular functions, including the immune response, apoptosis, cell proliferation, and inflammation. NFB is regulated by inhibitor pro- teins, IBs, which reside in the cytoplasm. The signal-induced phos- phorylation of the IBs by IKK- and -, and their subsequent ubiquitination-dependent degradation are prerequisite for NFB acti- vation (5–7). Activation of NFB has been shown to be critical for Raf-induced transformation (8). Raf-1 reportedly activates NFB by the induction of an autocrine loop in some cell types (9 –11), and it has been shown to induce NFB more directly in an MEK-independent, but MEK kinase-dependent manner (8). A common mechanism for protein kinase regulation is the phos- phorylation of one or more residues in the activation segment that is part of the catalytic site of many protein kinases. T598 and S601 are the major phosphorylation sites of B-Raf in response to oncogenic Ras, and phosphorylation of these two residues is required for full activation of B-Raf (12). These two residues, which are located within the kinase activation segment between kinase subdomains VII and VIII of B-Raf, are conserved in Raf-1. Furthermore, phosphorylation of T598 and S601 is important for B-Raf induction of Erk activation, Elk-1-dependent transcription, NIH 3T3 transformation and PC12 differentiation (12). The substitution of alanines for these residues reduces Ras-induced B-Raf activation; the replacement of these two sites by acidic amino acids results in the constitutive activation of kinase activity (12). Mutations in the B-Raf gene have been reported in a number of human cancers, including malignant melanoma, thyroid cancer, and colorectal carcinoma (13–22). B-Raf mutations occur in two regions of the B-Raf kinase domain, the glycine-rich loop and the activation segment. More than 80% of the mutations were T to A transversions at nucleotide 1796 (T1796A), leading to a substitution of glutamic acid for valine at amino acid 599 (V599E) in the activation segment. The V599E mutation is thought to mimic phosphorylation by inserting a negatively charged residue adjacent to the phosphorylation site at T598, rendering B-Raf constitutively active. The other three cancer- associated B-Raf mutants studied previously, i.e., another activation segment mutant (L596V) and two glycine-rich loop mutants (G463V and G468A), also had elevated kinase activity and NIH3T3-trans- forming activity as compared with those of wild-type B-Raf (13). Although several other B-Raf mutations were reported in previous studies (13–15), the biological effects of these mutations were not fully understood. In addition, the fact that the substitution of alanine for T598 or D593 reduced or abolished kinase and transforming activities prompted us to examine the possibility that some reported mutations in colorectal tumor such as D593V and T598I had no significant effect on tumorigenesis (12, 13, 15). However, the ob- served clustering of mutations within the activation segment, many at locations within one nucleotide of each other, would not appear to be the result of chance passenger mutations. In the present study, we demonstrate that colon cancer-associated B-Raf mutations within the kinase activation segment are not neces- sarily associated with an increase in Erk or NFB signaling activity, or in NIH3T3-transforming ability, suggesting that some reported Received 8/15/03; revised 10/17/03; accepted 10/20/03. Grant support: Program for the Organization for Pharmaceutical Safety and Research (OPSR), and the Japanese Ministry of Education, Culture, Sports, Science and Technol- ogy (MEXT). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Tsuneo Ikenoue, Division of Gastroenterology, The Institute for Adult Diseases, Asahi Life Foundation, 1-9-14, Nishi-Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. Phone: 81-3-3343-2151; Fax: 81-3-3344-6275; E-mail: t-ikenoue@ asahi-life.or.jp. 3 The abbreviations used are: Erk, extracellular signal-regulated kinase; IKK, inhibitor of nuclear factor B kinase; MEK, mitogen-activated protein/extracellular signal-regu- lated kinase kinase; Hsp, heat shock protein; MBP, myelin basic protein; NFB, nuclear factor B; IB, inhibitor of nuclear factor B; MAPK, mitogen-activated protein kinase; RIPA, radioimmunoprecipitation assay. 8132 Research. on November 26, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from