Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.

Whereas most kinesins motor along microtubules, KinI kinesins are microtubule depolymerizing machines. Surprisingly, we found that a KinI fragment consisting of only the motor core is capable of ATP-dependent depolymerization. The motor binds along microtubules in all nucleotide states, but in the presence of AMPPNP, microtubule depolymerization also occurs. Structural characterization of the products of AMPPNP-induced destabilization revealed a snapshot of the disassembly machine in action as it precisely deformed a tubulin dimer. While conventional kinesins use the energy of ATP binding to execute a "powerstroke," KinIs use it to bend the underlying protofilament. Thus, the relatively small class-specific differences within the KinI motor core modulate a fundamentally conserved mode of interaction with microtubules to produce a unique depolymerizing activity.

Hypoxia, reoxygenation, and the tyrosine phosphatase inhibitor pervanadate activate the transcription factor NF-B, involving phosphorylation of its inhibitor IB-␣ on tyrosine 42. This modification does not lead to degradation of IB by the proteasome͞ubiquitin pathway, as is seen on stimulation of cells with proinf lammatory cytokines. It is currently unknown how tyrosine-phosphorylated IB is removed from NF-B. Here we show that p85␣, the regulatory subunit of PI3-kinase, specifically associates through its Src homology 2 domains with tyrosine-phosphorylated IB-␣ in vitro and in vivo after stimulation of T cells with pervanadate. This association could provide a mechanism by which newly tyrosine-phosphorylated IB is sequestered from NF-B. Another mechanism by which PI3-kinase contributed to NF-B activation in response to pervanadate appeared to involve its catalytic p110 subunit. This was evident from the inhibition of pervanadate-induced NF-B activation and reporter gene induction by treatment of cells with nanomolar amounts of the PI3-kinase inhibitor wortmannin. The compound had virtually no effect on tumor necrosis factor-and interleukin-1-induced NF-B activities. Wortmannin did not inhibit tyrosine phosphorylation of IB-␣ or alter the stability of the PI3-kinase complex but inhibited Akt kinase activation in response to pervanadate. Our data suggest that both the regulatory and the catalytic subunit of PI3-kinase play a role in NF-B activation by the tyrosine phosphorylationdependent pathway.A large number of stimuli including proinflammatory cytokines, antigen stimulation of T and B cells, bacterial lipopolysaccharide, UV irradiation, viral infection, phorbol esters, and reactive oxygen intermediates can activate a family of transcription factors called NF-B͞Rel [for recent reviews see (1-3)]. The prototypical NF-B transcription factor is a heterodimer composed of p50 (NFB1) and p65 (RelA) DNAbinding subunits (4-9). Other members of this family in mammals include p52 (NFB2) (10, 11), RelB (12), c-Rel (13), p105 (4-7) and p100 (10, 11). These proteins share homology within an Ϸ300-amino acid domain termed the Rel homology domain, which mediates homo-and heterodimerization of the Rel family members as well as DNA-binding activity and nuclear localization (2, 3). Three members of the Rel family have a transcription activation domain (c-Rel, RelA, RelB) (14).The activity of NF-B is tightly controlled by a family of inhibitory proteins termed IB that sequester the transcription factor in the cytosol (2,3,14,15). IB proteins are structurally characterized by a cluster of 5 to 7 ankyrin repeats, a domain mediating the interaction with the Rel homology domain of NF-B͞Rel family members. Some NF-B stimuli trigger a cascade of events leading to the phosphorylation of IB, its polyubiquitination, and subsequent degradation by the 26S proteasome (16). Thereafter, the liberated NF-B is translocated to the nucleus and activates transcription of its many target genes. Among them are genes involved in the immune res...

Several members of the kinesin family of microtubule motor proteins play essential roles in mitotic spindle function and are potential targets for the discovery of novel antimitotic cancer therapies. KSP, also known as HsEg5, is a kinesin that plays an essential role in formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. We identified a potent inhibitor of KSP, CK0106023, which causes mitotic arrest and growth inhibition in several human tumor cell lines. Here we show that CK0106023 is an allosteric inhibitor of KSP motor domain ATPase with a K i of 12 nM. Among five kinesins tested, CK0106023 was specific for KSP. In tumor-bearing mice, CK0106023 exhibited antitumor activity comparable to or exceeding that of paclitaxel and caused the formation of monopolar mitotic figures identical to those produced in cultured cells. KSP was most abundant in proliferating human tissues and was absent from cultured postmitotic neurons. These findings are the first to demonstrate the feasibility of targeting mitotic kinesins for the treatment of cancer.

ReIA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-icB complex and contains a hh-affinity binding site for its cytoplasmic inhibitor, IkBa. After cellular activation, IKcBa is rapidly degraded in concert with the induced nuclear translocation of NF-ic. The present study demonstrates that tumor necrosis factor a-induced degradation of IicBa in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on IicBa result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (W) cytoplasmic ReIA is stably associated not only with IdcBa but also with other ankyrin motifrich proteins including the products of the NF-KB2 (plO0) and NF-ucBl (p105) genes; (it) in contrast to RelA-IhBa, ReIAp1OO cytoplasmic complexes are not dissociated following tumor necrosis factor a activation; (ii) p1O0 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p1O0 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal ofReIA, which is required for IucBa binding; (v) p1O0 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vW) as observed with IkcBa, nuclear RelA stimulates p1O0 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-icB.