The 7-methyl guanosine cap structure of RNA is essential for key aspects of RNA processing, including pre-mRNA splicing, 3' end formation, U snRNA transport, nonsense-mediated decay and translation. Two cap-binding proteins mediate these effects: cytosolic eIF-4E and nuclear cap-binding protein complex (CBC). The latter consists of a CBP20 subunit, which binds the cap, and a CBP80 subunit, which ensures high-affinity cap binding. Here we report the 2.1 A resolution structure of human CBC with the cap analog m7GpppG, as well as the structure of unliganded CBC. Comparisons between these structures indicate that the cap induces substantial conformational changes within the N-terminal loop of CBP20, enabling Tyr 20 to join Tyr 43 in pi-pi stacking interactions with the methylated guanosine base. CBP80 stabilizes the movement of the N-terminal loop of CBP20 and locks the CBC into a high affinity cap-binding state. The structure for the CBC bound to m7GpppG highlights interesting similarities and differences between CBC and eIF-4E, and provides insights into the regulatory mechanisms used by growth factors and other extracellular stimuli to influence the cap-binding state of the CBC.

Opposing cellular responses are typically regulated by distinct sets of genes. However, tissue transglutaminase (TGase) provides an interesting example of a single gene product that has been implicated both in affording protection against cellular insults as well as in promoting cell death. Here, we shed some light on how these conflicting activities might be manifested by demonstrating that alternative transcripts of TGase differentially affect cell viability. We show that although the full-length TGase protein affords strong protection against cell death signals, a shorter version of TGase that is truncated at the 3 end, and thus called TGase-short (TGase-S), is cytotoxic. The apoptotic activity of TGase-S is not dependent on its transamidation activity because the mutation of a cysteine residue that is essential for catalyzing this reaction does not compromise the ability of TGase-S to induce cell death. Intriguingly, TGase-S undergoes inappropriate oligomer formation in cells before cell death, suggesting a novel mechanism for the apoptotic effects of this protein.aggregation ͉ cell death ͉ transamidation

Cool-1 (cloned-out of library 1) has a key role in regulating epidermal growth factor receptor (EGFR) degradation. Here, we show that Cool-1 performs this function by functioning as both an upstream activator and downstream target for Cdc42. EGF-dependent phosphorylation of Cool-1 enables it to act as a nucleotide exchange factor for Cdc42 and to form a complex with the E3 ligase Cbl, thus regulating Cbl-catalysed EGFR degradation. The EGF-dependent phosphorylation is normally transient; however, Cool-1 phosphorylation is sustained in cells expressing v-Src and is essential for cellular transformation, as well as for v-Src-induced tumour formation in mice. These findings demonstrate that the regulated phosphorylation of Cool-1 is necessary to maintain the balance between normal signalling by EGFR and Src versus aberrant growth and transformation.

Although the small GTPase Ran is best known for its roles in nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation, recent studies have demonstrated the overexpression of Ran in multiple tumor types and that its expression is correlated with a poor patient prognosis, providing evidence for the importance of this GTPase in cell growth regulation. Here we show that Ran is subject to growth factor regulation by demonstrating that it is activated in a serum-dependent manner in human breast cancer cells and, in particular, in response to heregulin, a growth factor that activates the Neu/ ErbB2 tyrosine kinase. The heregulin-dependent activation of Ran requires mTOR (mammalian target of rapamycin) and stimulates the capped RNA binding capability of the cap-binding complex in the nucleus, thus influencing gene expression at the level of mRNA processing. We further demonstrate that the excessive activation of Ran has important consequences for cell growth by showing that a novel, activated Ran mutant is sufficient to transform NIH-3T3 cells in an mTOR-and epidermal growth factor receptor-dependent manner and that Ran-transformed cells form tumors in mice.Ran is a unique member of the Ras superfamily of GTPases that utilizes a guanine nucleotide exchange factor (the chromatin-associated RCC1 protein), a GTPase-accelerating protein complex (RanGAP/RanBP1), and a single major class of effectors (the importins or karyopherins) to regulate the nucleocytoplasmic transport of various cargo as well as other nuclear functions (for review, see Ref. 1). In interphase cells, a Ran-GTP gradient is formed in response to the nuclear localization of RCC1 together with the cytoplasmic localization of RanGAP. As a result, Ran exists predominantly in an active, GTP-bound state in the nucleus where it is capable of engaging its primary biological effectors, the importins/karyopherins. GTP hydrolysis, catalyzed by RanGAP in the cytoplasm, then results in the dissociation of Ran-GDP from its effector proteins. The nucleocytoplasmic transport of a number of proteins is dependent upon the proper establishment of the Ran-GTP gradient, as best exemplified in the case of classical nuclear import (2). Protein cargo destined for the nucleus is identified by the presence of a nuclear localization sequence. The nuclear localization sequence is recognized by an adapter protein, importin-␣, in the cytosol, and upon binding the cargo, importin-␣ engages importin-␤ to form a complete import complex that translocates to the nucleus. Within the nucleus, Ran-GTP binds to importin-␤, causing it to dissociate from the import complex, which results in the subsequent release of cargo. Thus, the fact that Ran-GTP is predominantly a nuclear species is pivotal in the directional release of import cargo into the nucleus.Similarly, it has been suggested that Ran plays an essential role in the directional release of capped RNAs in the cytoplasm by regulating the interactions that occur between the nuclear cap-binding complex (CBC) 2 ...

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