Identification of cisplatin-binding proteins using agarose conjugates of platinum compounds.

Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

Precipitation of kidney myosin IIA and IIB by freezing.

Actomyosin precipitation is a critical step in the purification of myosins. In this work, the objective was to precipitate rat kidney actomyosin and isolate myosin by freezing and thawing the soluble fraction. Kidney was homogenized in imidazole buffer, centrifuged at 45000 g for 30 min, and the supernatant was frozen at -20°C for 48 h. The supernatant was thawed at 4°C, centrifuged at 45000 g for 30 min and the precipitate washed twice with imidazole buffer pH 7.0 (with and without Triton X-100, respectively). The resulting precipitate presented a polypeptide profile in SDS/PAGE characteristic of actomyosin and expressed Mg- and K/EDTA-ATPase activity. The actomyosin complex was solubilized with ATP and Mg, and the main polypeptide, p200, was purified in a DEAE-Sepharose column. p200 was marked with anti-myosin II, co-sedimented with F-actin in the absence, but not in the presence, of ATP and was identified by MS/MS with a high Mascot score for myosin IIA. The analysis identified peptides exclusive of myosin IIB, but detected no peptides exclusive of myosin IIC.

Menin, a tumor suppressor, associates with nonmuscle myosin II-A heavy chain.

MEN1 is a likely tumor suppressor gene that encodes a novel protein, menin. Menin is a 610 amino-acid residue protein with as yet unknown function(s). We have used tandem affinity purification and mass spectroscopy to isolate and identify proteins associating with menin from cultured HeLa cell extracts. This strategy has resulted in the isolation and identification of nonmuscle myosin type II-A heavy chain (NMHC II-A) as a menin interacting protein. This interaction was confirmed by glutathione-S-transferase pulldown assays, by coimmunoprecipitation, and by actin selection of myosin. We have further identified the amino-terminal region of menin and the head domain of NMHC II-A to be regions required for this interaction. Moreover menin was seen to colocalize with this myosin isoform in the cleavage furrow of dividing cells by indirect immunofluoresence. These data indicate that menin through binding to NMHC II-A could participate in cell division and in other processes that involve NMHC II-A.