Atomic structure of the KEOPS complex: an ancient protein kinase-containing molecular machine.

Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.

Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays.

The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be yen-[Ile/Leu/Val]-Ser/Thr-phi-phi-X- yen/Pro (where phi denotes a large hydrophobic residue, yen is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.

Identification of a novel c-Myc protein interactor, JPO2, with transforming activity in medulloblastoma cells.

c-myc oncogene activation is critical in the pathogenesis of a spectrum of human malignancies. The c-Myc NH2-terminal domain (MycNTD) is essential for cellular transformation, and mediates critical protein interactions that modulate c-Myc oncogenic properties. In medulloblastoma, the most common malignant pediatric brain tumor, deregulated c-myc expression is linked with poorer disease phenotypes and outcomes. The biological basis for these associations is, however, not well understood. To better understand mechanisms underlying Myc-mediated transformation of medulloblastoma, we sought to identify novel MycNTD protein interactors from a medulloblastoma cell line library using a unique two-hybrid system. We identified a novel MycNTD binding protein, JPO2, which shows nuclear colocalization with c-Myc, and interacts with c-Myc both in vitro and in mammalian cells. In Rat1a transformation assays, JPO2 potentiates c-Myc transforming activity, and can complement a transformation-defective Myc mutant. Immunohistochemical studies indicate tumor-specific JPO2 expression in human medulloblastoma, and an association of JPO2 expression with metastatic tumors. Significantly, JPO2 expression induces colony formation in UW228, a medulloblastoma cell line, whereas RNAi-mediated JPO2 knockdown impairs colony formation in UW228, and in Myc-transformed UW228 cells. These data provide evidence for biochemical and functional interaction between c-Myc and JPO2 in medulloblastoma transformation. JPO2 is closely related to JPO1, a Myc transcriptional target with transforming activity. As tumor-specific JPO1 expression in human and murine medulloblastoma has also been reported; these collective observations suggest important functional links between the novel JPO protein family and c-Myc in medulloblastoma transformation.

A structure-based model of the c-Myc/Bin1 protein interaction shows alternative splicing of Bin1 and c-Myc phosphorylation are key binding determinants.

The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and apoptosis can be modulated by the expression of Bin1. Multiple splicing of the Bin1 transcript results in ubiquitous, tissue-specific and tumor-specific populations of Bin1 proteins in vivo. We report on the structural features of the interaction between c-Myc and Bin1, and describe two mechanisms by which the binding of different Bin1 isoforms to c-Myc may be regulated in cells. Our findings identify a consensus class II SH3-binding motif in c-Myc and the C-terminal SH3 domain of Bin1 as the primary structure determinants of their interaction. We present biochemical and structural evidence that tumor-specific isoforms of Bin1 are precluded from interaction with c-Myc through an intramolecular polyproline-SH3 domain interaction that inhibits the Bin1 SH3 domain from binding to c-Myc. Furthermore, c-Myc/Bin1 interaction can be inhibited by phosphorylation of c-Myc at Ser62, a functionally important residue found within the c-Myc SH3-binding motif. Our data provide a structure-based model of the c-Myc/Bin1 interaction and suggest a mode of regulation that may be important for c-Myc function as a regulator of gene transcription.

Promoter-binding and repression of PDGFRB by c-Myc are separable activities.

The c-Myc transcription factor represses the mRNA expression of the platelet-derived growth factor receptor beta gene (PDGFRB). Using chromatin immunoprecipitation, we show that c-Myc binds to the proximal promoter of the PDGFRB gene in proliferating rat fibroblasts. Interestingly, mutant c-Myc proteins that are unable to repress PDGFRB gene expression, c-Myc(dBR) and c-Myc(d106-143), are still able to bind to the promoter in vivo. Hence, promoter-binding and repression of PDGFRB by c-Myc are separable activities. We also show that Myc repression of PDGFRB is not dependent on previously described or known transactivator-binding regions, suggesting Myc may be recruited to the promoter by multiple or yet unidentified transcription factors. In the presence of intact promoter-binding by Myc, trichostatin A (TSA) can block Myc repression of PDGFRB in vivo, again demonstrating that promoter-binding and repression are separable. Taken together, we hypothesize that Myc repression of PDGFRB expression occurs by a multi-step mechanism in which repression is initiated after Myc is recruited to the promoter.

The myc oncogene: MarvelouslY Complex.

The activated product of the myc oncogene deregulates both cell growth and death check points and, in a permissive environment, rapidly accelerates the affected clone through the carcinogenic process. Advances in understanding the molecular mechanism of Myc action are highlighted in this review. With the revolutionary developments in molecular diagnostic technology, we have witnessed an unprecedented advance in detecting activated myc in its deregulated, oncogenic form in primary human cancers. These improvements provide new opportunities to appreciate the tumor subtypes harboring deregulated Myc expression, to identify the essential cooperating lesions, and to realize the therapeutic potential of targeting Myc. Knowledge of both the breadth and depth of the numerous biological activities controlled by Myc has also been an area of progress. Myc is a multifunctional protein that can regulate cell cycle, cell growth, differentiation, apoptosis, transformation, genomic instability, and angiogenesis. New insights into Myc's role in regulating these diverse activities are discussed. In addition, breakthroughs in understanding Myc as a regulator of gene transcription have revealed multiple mechanisms of Myc activation and repression of target genes. Moreover, the number of reported Myc regulated genes has expanded in the past few years, inspiring a need to focus on classifying and segregating bona fide targets. Finally, the identity of Myc-binding proteins has been difficult, yet has exploded in the past few years with a plethora of novel interactors. Their characterization and potential impact on Myc function are discussed. The rapidity and magnitude of recent progress in the Myc field strongly suggests that this marvelously complex molecule will soon be unmasked.

Structural basis of Rad53 kinase activation by dimerization and activation segment exchange.

The protein kinase Rad53 is a key regulator of the DNA damage checkpoint in budding yeast. Its human ortholog, CHEK2, is mutated in familial breast cancer and mediates apoptosis in response to genotoxic stress. Autophosphorylation of Rad53 at residue Thr354 located in the kinase activation segment is essential for Rad53 activation. In this study, we assessed the requirement of kinase domain dimerization and the exchange of its activation segment during the Rad53 activation process. We solved the crystal structure of Rad53 in its dimeric form and found that disruption of the observed head-to-tail, face-to-face dimer structure decreased Rad53 autophosphorylation on Thr354 in vitro and impaired Rad53 function in vivo. Moreover, we provide critical functional evidence that Rad53 trans-autophosphorylation may involve the interkinase domain exchange of helix αEF via an invariant salt bridge. These findings suggest a mechanism of autophosphorylation that may be broadly applicable to other protein kinases.