Biochemistry
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Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. ⋯ The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.
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Comparative Study
Localization of the antimitotic peptide and depsipeptide binding site on beta-tubulin.
Several naturally occurring peptides and depsipeptides which include the cryptophycins, dolastatin 10, hemiasterlin, and phomopsin A have been found to be potent antimitotic agents, causing cell death at picomolar or low nanomolar concentrations. These compounds inhibit microtubule growth, modulate the dynamics of microtubules, and induce the self-association of tubulin dimers into single-walled rings and spirals. These peptides exhibit mutual competitive inhibition in binding to beta-tubulin, while noncompetitively inhibiting the binding of vinblastine and vincristine to beta-tubulin. ⋯ Application of these same methods to alpha-tubulin indicated no interaction between alpha-tubulin and any of the peptides. On the basis of the docking results, a model for the mechanism of microtubule disruption and formation of aberrant nonmicrotubule structures is proposed. Both the active site and mechanism of microtubule depolymerization predictions are in good agreement with experimental findings.
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Ecteinascidin 743 (Et743) is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC-rich sequences resulting in an unusual bending toward the major groove. The ability of Et743 to block DNA replication was studied using the well-established simian virus (SV40) model for mammalian DNA replication in cells and cell-free extracts. Intracellular SV40 DNA isolated from Et743-treated BSC-1 cells was analyzed by native, two-dimensional agarose gel electrophoresis. ⋯ Comparative studies involving related DNA alkylators, tomamycin and saframycin A, revealed inhibition of SV40 DNA replication in cells at concentrations approximately 10 times higher than Et743. Under cell-free conditions tomamycin- or saframycin-A-adducted DNA templates inhibited DNA synthesis similarly to Et743. Et743 appears to be unusual among other alkylators, because its adducts strongly inhibit intracellular SV40 DNA replication but are relatively weak as cis inhibitors as measured under cell-free conditions.