A Link between Increased Transforming Activity of Lymphoma-Derived MYC Mutant Alleles, Their Defective Regulation by p107, and Altered Phosphorylation of the c-Myc Transactivation Domain

Abstract

The c-Myc protein is a transcription factor with an N-terminal transcriptional regulatory domain and C-terminal oligomerization and DNA-binding motifs. Previous studies have demonstrated that p107, a protein related to the retinoblastoma protein, binds to the c-Myc transcriptional activation domain and suppresses its activity. We sought to characterize the transforming activity and transcriptional properties of lymphomaderived mutant MYC alleles. Alleles encoding c-Myc proteins with missense mutations in the transcriptional regulatory domain were more potent than wild-type c-Myc in transforming rodent fibroblasts. Although the mutant c-Myc proteins retained their binding to p107 in in vitro and in vivo assays, p107 failed to suppress their transcriptional activation activities. Many of the lymphoma-derived MYC alleles contain missense mutations that result in substitution for the threonine at codon 58 or affect sequences flanking this amino acid. We observed that in vivo phosphorylation of Thr-58 was absent in a lymphoma cell line with a mutant MYC allele containing a missense mutation flanking codon 58. Our in vitro studies suggest that phosphorylation of Thr-58 in wild-type c-Myc was dependent on cyclin A and required prior phosphorylation of Ser-62 by a p107-cyclin A-CDK complex. In contrast, Thr-58 remained unphosphorylated in two representative mutant c-Myc transactivation domains in vitro. Our studies suggest that missense mutations in MYC may be selected for during lymphomagenesis, because the mutant MYC proteins have altered functional interactions with p107 protein complexes and fail to be phosphorylated at Thr-58.The MYC proto-oncogene encodes a helix-loop-helixleucine zipper (HLH-Z) transcription factor that participates in the regulation of cell proliferation, cell differentiation, and apoptosis (13,17,18,21,43,58). Deregulation of MYC expression appears to be involved in the pathogenesis of a variety of human neoplasms. Two regions of the Myc protein required for its biological activities are the N-terminal transcription activation domain (TAD) and the C-terminal basic-HLH-Zspecific DNA-binding domain (18,67). The HLH-Z domain mediates dimerization of Myc with its partner, Max, and aligns the adjacent basic regions in the major groove of DNA for recognition of the specific E-box sequence, CAC(A/G)TG (2, 3, 7, 10-13, 17, 29, 44, 46, 50, 57, 58, 69). The TAD of Myc has been shown to activate transcription in the chimeric GAL4 system as well as through E-box sequences with full-length Myc (6,33,42,46,55,62,63). Myc may also interact with TFII-I and impede transcription that is dependent on the initiator (Inr) element (49,55,62,63). Although the Myc TAD has been shown to contact the TATA-binding protein (TBP), the mechanisms by which Myc regulates transcription in normal and neoplastic cells remain poorly understood (36, 52).Clues to the importance of the Myc N-terminal TAD in neoplastic transformation were recognized through studies of Myc mutants and related TADs of L-Myc and B-Myc (4, 61). L-Myc, wh...

MYC oncogenes and human neoplastic disease

“…This appears to be accomplished without any alteration in the rate of c-myc-induced apoptosis (Hoang et al, 1995).…”

“…Despite the unresolved controversy surrounding the roles of Thr 58 and Ser 62 in transactivation, most investigators are in agreement that both engineered and BL-derived mutations of these regions possess increased transforming activity (Henriksson et al, 1993;Pulverer et al, 1994;Hoang et al, 1995). This appears to be accomplished without any alteration in the rate of c-myc-induced apoptosis (Hoang et al, 1995).…”

“…They showed that the transactivation and transformation functions of wildtype c-myc are normally subject to down-regulation by the pRB-related protein p107 as the result of a direct physical interaction between the two proteins. In contrast, many of the c-myc mutants from BL tumors have lost the ability to be down-regulated by p107, although association between the two proteins still occurs (Hoang et al, 1995). A more recent study, however, indicated that loss of p107-mediated negative regulation of c-myc transactivation is not an inevitable result of c-myc mutation (Smith-Sorenson et al, 1996).…”

Mechanisms of apoptosis by c-Myc

“…A good candidate, however, has emerged in the ATM-related protein TRRAP, an NTD-binding protein that is part of the SAGA complex which includes histone deacetylases implicated in activation events (Grant et al, 1998;McMahon et al, 1998;Saleh et al, 1998). The activation activity of the NTD may also be influenced by interactions with the retinoblastoma (Rb)-related protein p107 (Beijersbergen et al, 1994;Gu et al, 1994;Hoang et al, 1995), whose binding to c-Myc may be modulated by cyclin D/CDK4 phosphorylation (Hass et al, 1997), or with the adaptor protein and tumor suppressor Bin1 (Sakamuro et al, 1996;Elliott et al, 1999). The NTD is also reported to interact with a-tubulin (Alexandrova et al, 1995), PAM, a large protein which includes RCC1-like repeats suggesting a role in chromatin regulation , MM-1, a nucleocytoplasmic adaptor protein that inhibits transactivation by c-Myc, and AMY-1, a small protein reported to potentiate the transactivation activity of cMyc (Taira et al, 1998).…”

Retinoblastoma gene family expression in lymphoid tissues

“…In addition, reports showing that the pRb-related protein pRb2/p130 is mutated in its nuclear localization signal in BL lines and primary tumours raise the possibility that inactivation of this member of the pRB family renders BL cells more susceptible to transformation by activated c-Myc (Cinti et al, 2000). Interaction between p107 and c-myc has been demonstrated and during lymphomagenesis missense mutations of c-Myc may be selected because the mutant Myc protein has altered interactions with p107 (Hoang et al, 1995).…”