The N-Terminal Domain of c-Myc Associates with α-Tubulin and Microtubules In Vivo and In Vitro

Abstract

The polymerization of ␣-and ␤-tubulin into microtubules results in a complex network of microfibrils that have important structural and functional roles in all eukaryotic cells. In addition, microtubules can interact with a diverse family of polypeptides which are believed to directly promote the assembly of microtubules and to modulate their functional activity. We have demonstrated that the c-Myc oncoprotein interacts in vivo and in vitro with ␣-tubulin and with polymerized microtubules and have defined the binding site to the N-terminal region within the transactivation domain of c-Myc. In addition, we have shown that c-Myc colocalizes with microtubules and remains tightly bound to the microtubule network after detergent extraction of intact cells. These findings suggest a potential role for Myc-tubulin interaction in vivo.The product of the c-myc gene is a nuclear phosphoprotein that has been implicated in the regulation of cell differentiation, apoptosis, and the development of human tumors (5,19,28). The c-Myc protein includes multifunctional domains that include an N-terminal transactivation domain within exon II and a C-terminal domain, within exon III, which is necessary for heterodimerization (5, 19). These observations have supported the model that c-Myc functions as a transcription factor whose activity can be regulated by its protein binding partners. In addition, the N-terminal transactivation region of c-Myc binds to the retinoblastoma protein-related tumor suppressor protein p107, and it has been recently shown that this interaction can suppress the activity of the c-Myc transactivation domain (12). Further, the observation that Burkitt's lymphoma frequently contains naturally occurring somatic mutations within the transactivation domain (3, 34) which result in loss of p107 suppression confirms that this is an important functional domain of c-Myc.Although c-Myc is characterized as a nuclear protein (10, 15), it has recently been proposed that the subcellular localization of c-Myc can vary according to the proliferation state of the cell. For example, in actively growing NIH 3T3 cells c-Myc is predominantly found in the nucleus, while a shift to the cytoplasm occurs in contact-inhibited cells (31). An increased cytoplasmic/nuclear ratio for c-Myc has also been noted following monocytic differentiation of human myeloid leukemia cells (8) and in nondividing prefertilized Xenopus oocytes (14). In addition, comparison of c-Myc localization patterns of normal tissue, adenomas, and colorectal tumors showed that tumor progression was associated with an accumulation of cytoplasmic c-Myc protein (27). These experiments suggested that nucleus-cytoplasm exchanges of c-Myc protein play an important functional role in cell proliferation. Since it had been proposed that tubulin mediates the cytoplasm-to-nucleus translocation of the glucocorticoid and vitamin D receptors (2, 25), we examined whether c-Myc could also interact with tubulin and microtubules. This hypothesis was also suggested by the observation tha...

Mechanisms of apoptosis by c-Myc

“…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). In addition to harboring a transactivation domain, the NTD also mediates transcriptional repression (Kaddurah-Daouk et al, 1987;Suen and Hung, 1991;Yang et al, 1991Yang et al, , 1993Jansen-Durr et al, 1993;Roy et al, 1993;Li et al, 1994;Philipp et al, 1994;Lee et al, 1996;Tikhonenko et al, 1997).…”

MSSP promotes ras/myc cooperative cell transforming activity by binding to c‐Myc

“…Complex formation with various proteins may realize versatile functions of c-Myc, and indeed several proteins have been identi®ed as associating with c-Myc. Max (Blackwood & Eisenman 1991;Blackwood et al 1992), Nmi (Bao & Zervos 1996), YY1 (Shrivastava et al 1993) AP2 (Gaubatz et al 1995), SNF5 (Cheng et al 1999) and CBF/NF-Y (Taira et al 1999) bind to the C-terminal region of c-Myc, while p107 (Beijersbergen et al 1994;Gu et al 1994;Hoang et al 1995), Bin1 (Sakamuro et al 1996), TBP (Hateboer et al 1993;Maheswaran et al 1994), a-tubulin (Alexandrova et al 1995), TRRAP (McMahon et al 1998), Pam (Guo et al 1998), AMY-1 ) and MM-1 (Mori et al 1998) bind to the N-terminal region. Max stimulates the activities of c-Myc in transcription and transformation by heterodimer formation via a leucine zipper (Blackwood & Eisenman 1991;Blackwood et al 1992).…”

c‐Raf kinase binds to N‐terminal domain of c‐Myc

“…Cell extract (200 lal, containing 100, gg of total protein) was incubated with GST fusion proteins prebound to 20 btl of glutathione-agarose beads as described [10]. After 60 min of incubation at 4°C the agarose beads were washed 5 times.…”

“…Construction of pGEX-based plasmids (Pharmacia), containing GST Myc fusion proteins, bearing a unique thrombin cleavage site between Myc and GST moieties, have been described [10].…”

“…In addition, MAP kinase has been shown to bind and phosphorylates c-Myc in this region [9]. In our previous work, we showed, that c-Myc N-terminal region is able to bind to tubulin and to microtubules [10].…”