No abstract

S100A4, also known as mts1, is a member of the S100 family of Ca 2+ -binding proteins that is directly involved in tumor invasion and metastasis via interactions with specific protein targets, including nonmuscle myosin-IIA (MIIA). Human S100A4 binds two Ca 2+ ions with the typical EF-hand exhibiting an affinity that is nearly 1 order of magnitude tighter than that of the pseudo-EF-hand. To examine how Ca 2+ modifies the overall organization and structure of the protein, we determined the 1.7 Å crystal structure of the human Ca 2+ -S100A4. Ca 2+ binding induces a large reorientation of helix 3 in the typical EF-hand. This reorganization exposes a hydrophobic cleft that is comprised of residues from the hinge region, helix 3, and helix 4, which afford specific target recognition and binding. The Ca 2+ -dependent conformational change is required for S100A4 to bind peptide sequences derived from the C-terminal portion of the MIIA rod with submicromolar affinity. In addition, the level of binding of Ca 2+ to both EF-hands increases by 1 order of magnitude in the presence of MIIA. NMR spectroscopy studies demonstrate that following titration with a MIIA peptide, the largest chemical shift perturbations and exchange broadening effects occur for residues in the hydrophobic pocket of Ca 2+ -S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca 2+ dependence of formation of the S100A4-MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function. S100A4, also called mts1, is a member of the S100 family of small, homodimeric, EF-hand Ca 2+ binding proteins. S100 proteins are expressed in a tissue specific manner and bind to a variety of target proteins, resulting in the regulation of specific cellular processes, including cell-cycle regulation, protein phosphorylation, cell growth, motility, differentiation, and survival (1-4). While S100A4 is expressed in a wide range of normal tissues (5,6), it is recognized that an increased level of S100A4 expression correlates with a high incidence of metastasis and poor prognosis for cancer patients (7,8). High S100A4 expression levels are associated with several metastatic cancers, including breast (9), colorectal (10), bladder (11), † This work was supported by National Institutes of Health Grants GM069945 (A.R.B.), GM58888 (D.J.W.), and CA107331 (D.J. W.) and American Cancer Society Grant CDD107745 (D.J.W.). * To whom correspondence should be addressed: A.R.B.: telephone, (718) 430-2741; fax, (718) 430-8565; e-mail, bresnick@aecom.yu.edu. D.J.W.: telephone, (410) 706-4354; fax, (410) 706-0458; e-mail, dweber@umaryland.edu. S.C.A.: telephone, (718) 430-2746; fax, (718) 430-8565; e-mail, almo@aecom.yu.edu. § These authors contributed equally to the completion of this work. ⊥ Current address: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K. NIH Public Access Author ManuscriptBiochemistry. Auth...

Bipolar spindle formation is essential for the accurate segregation of genetic material during cell division. Although centrosomes influence the number of spindle poles during mitosis, motor and non-motor microtubule-associated proteins (MAPs) also play key roles in determining spindle morphology. TPX2 is a novel MAP also characterized in Xenopus cell-free extracts. To examine hTPX2 (human TPX2) function in human cells, we used siRNA to knock-down its expression and found that cells lacking hTPX2 arrest in mitosis with multipolar spindles. NuMA, gamma-tubulin, and centrin localize to each pole, and nocodazole treatment of cells lacking hTPX2 demonstrates that the localization of gamma-tubulin to multiple spindle poles requires intact microtubules. Furthermore, we show that the formation of monopolar microtubule arrays in human cell extracts does not require hTPX2, demonstrating that the mechanism by which hTPX2 promotes spindle bipolarity is independent of activities focusing microtubule minus ends at spindle poles. Finally, inhibition of the kinesin Eg5 in hTPX2-depleted cells leads to monopolar spindles, indicating that Eg5 function is necessary for multipolar spindle formation in the absence of hTPX2. Our observations reveal a structural role for hTPX2 in spindles and provide evidence for a balance between microtubule-based motor forces and structural spindle components.

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.