Myc proteins in brain tumor development and maintenance 125 hindbrain (5), while c-myc is expressed at lower levels (6,8). In normal cerebellar granule cells, Mycn is important for cell replication, whereas Mycn protein degradation is essential for cell cycle exit (69–71). Importantly, Mycn depletion in nestin-driven NSCs reduces both germinal zones of the developing cere- bellum, including the neuroepithelial cells lining the fourth ventricle as well as the rhombic lip structure with the granule precursor cells forming the external germinal layer (5). The depletion also leads to dis- rupted neuronal differentiation which correlates with the ﬁndings that Myc genes (including c-myc, Mycn, and Mycl) promote (but are not required for (72)) the dedifferentiation processes for producing induced pluripotent stem (iPS) cells (73,74). High expression of Myc proteins, at least when using v-myc (the retroviral homolog of c-myc) transduction, is impor- tant for immortalization and self-renewal of NSCs (75,76). Interestingly, Mad proteins also appear to have a role in neuronal differentiation (77). Mxi1, another member of the Mad family of proteins, can antagonize Myc oncoproteins in vivo (78). Overex- pression of Mxi1 in human glioma cell lines reduces their mitotic activity (79), possibly through down- regulation of cyclin B1 expression (80). The data suggest that Mxi1 that interacts with Max can antagonize Myc and thereby suppress glioma progression. Myc protein stability Myc proteins are extremely unstable with half-lives of only 20–30 min (81). There are two phosphorylation sites on Myc proteins which are primarily responsible for stabilization (Figure 1). First, serine (S62) is phosphorylated which leads to Myc stabilization (82). The extracellular-regulated kinase 1, 2 (ERK) is known to mediate S62 phosphorylation (83). How- ever, in cerebellar neural precursors, the mitotic cyclin-dependent kinase 1 (Cdk1) is the priming kinase for Mycn at S62 (70). Following S62 phos- phorylation of Myc, threonine 58 (T58) is phospho- rylated by glycogen synthase kinase 3 beta (GSK3b) which will target Myc for degradation (84). Under normal conditions T58 is always phosphorylated after S62. The turnover of Myc is mostly controlled by ubiquitin-mediated proteolysis (85,86), which targets the speciﬁc Myc proteins with great precision. Speciﬁc ubiquitin-protein ligases (of type E3) like Fbw7 can recognize and bind to Myc, but only when T58 is phosphorylated alone. This requires dephosphorylation of S62 with the help of a prolyl isomerase, Pin1, that binds the phosphorylated sites and isomerizes Myc on proline 59 (87). This provides a conformation change of Myc which enables protein phosphatase 2A (PP2A) to remove the phosphate group. Fbw7 can now recognize Myc with a single phosphorylation on T58 and send Myc for proteolytic degradation (88) by ubiquitylating Myc on its amino- terminus. It is suggested that at least four ubiquitin groups need to be linked to a substrate in order for it to be recognized by a proteasome that will later dissect it into small peptide fragments (89). Increased stability of Myc proteins can explain the reported inconsistency between gene ampliﬁcations and protein levels of Myc found in solid tumors, like in breast cancer (90,91). There are numerous reports of enhanced expression levels of Myc oncoproteins in many different types of tumors, that suggesting this is an important event for tumor initiation or an apparent advantage for tumor progression (reviewed in (92)). Myc protein levels are elevated in subtypes of glioma (14), but there are few reports in which a correlation between expression/ampliﬁcation and protein levels of Myc proteins is assessed. Such a relationship would be required in order to understand fully the extent of putative Myc protein stabilization in these brain tumors. Reports of mutations or alterations in genes that can regulate Myc protein stability are increasing (for a recent review, see (93)). For example, Fbw7 can keep Myc levels low and therefore act as a tumor suppressor. In the absence of Fbw7, the levels of Myc and the activity of the protein will increase (94), and mutations in Fbw7 prevent Myc degradation in T-cell acute lymphoblastic leukemia (T-ALL) (95). With regard to the discussions of how brain tumor develops it is interesting to ﬁnd that Fbw7 is highly expressed during normal brain development (96,97), where it can possibly regulate Myc protein stability. Other ubiquitin ligases recognize Myc on other sites than Fbw7 does, like S phase kinase- associated protein 2 (Skp2). Skp2 is responsible for Myc protein turnover (98) but is paradoxically also promoting Myc activity by functionally amplifying the Myc response (99). How Skp2 is working like an oncogene and how it correlates with Myc is not fully known (93), but Skp2 is actually associating with Myc target genes when Myc is active (100). Other proteins involved in regulating stability of Myc proteins are also mutated in cancer, like Usp28, which has been found frequently up-regulated in colon adenocarci- nomas (101). This soluble deubiquitylating enzyme (DUB) stabilizes Myc by removing linked ubiquitin chains that have been conjugated by Fbw7 (Figure 1). TRIM32 is another ubiquitin ligase that is asymmet- rically distributed during NSC division and is inhibiting cell proliferation by promoting Myc degradation and neuronal differentiation (102).