ABSTRACT
Dynactin is a multi-subunit complex that has been implicated in the function of cytoplasmic dynein which is a minus end - directed microtubule motor protein with numerous functions including nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during interphase and mitosis. Dynamitin,the 50 kD subunit of dynactin, is important for stabilizing the dynactin complex. To gain more insight into the mechanism of stabilizing, we analyzed the sequence of dynamitin and revealed that domains of dynamitin is homology to the Walker A and Walker B ATPase motifs. The purified GST-dynamitin and GST-free dynamitin both showed ATPase activity specifically. An inactivating mutation in the Walker A, but not the Walker B ATPase motif abolished the ATPase activity of dynamitin. The mutational analysis studies further supported that dynamitin is an ATPase. Kinetic studies of the ATPase activity of dynamitin revealed a Km for ATP of 125.78μmol/L and a kcat of 7.4 min-1.
ABSTRACT
Aim To study the mechanism of inhibition of basic fibroblast growth factor (bFGF) related signal transduction by lidamycin in cancer cells. Methods MTT assay was used to determine the growth inhibitory effect of lidamycin (LDM) and adriamycin (ADR) in several cancer cell lines. The inhibition of bFGF bound to its receptor by LDM was measured with [ 125I ]-bFGF binding assay.Intracellular Ca2+ stimulated by bFGF was measured by Fura-3. The formation of bFGF- receptor immune complex and the inhibitory effect of LDM on the activity of PKC isoenzymes induced by bFGF in cancer cells were identified by Western blotting analysis. Results LDM displayed extremely potent growth inhibitory effect on several cancer cell lines in a dose-dependent manner. A comparison of the IC50 values showed that the effect of LDM was 1 000-fold more potent than that of ADR. LDM blocked the specific by anti-FGFR specific antibody, LDM inhibited the formation of bFGF-receptor immune complex. bFGF demonstrated that LDM inhibited the activity of PKC isoenzymes in cancer cells stimulated with bFGF.Conclusion The blocking of bFGF receptors in the signal transduction pathway may be involved in the effect of LDM on cancer cells.