Tanimura et al. demonstrate that SH3P2 binds to and functions as a cytosolic anchor for myosin 1E (Myo1E). ERK signaling–dependent phosphorylation of SH3P2 induces the dissociation of bound Myo1E and its consequent localization to the tips of lamellipodia, where it promotes cell motility.
The 70-kDa heat shock protein (Hsp70) is up-regulated in a wide variety of tumor cell types and contributes to the resistance of these cells to the induction of cell death by anticancer drugs. Hsp70 binding protein 1 (HspBP1) modulates the activity of Hsp70 but its biological significance has remained unclear. We have now examined whether HspBP1 might interfere with the prosurvival function of Hsp70, which is mediated, at least in part, by inhibition of the death-associated permeabilization of lysosomal membranes. HspBP1 was found to be expressed at a higher level than Hsp70 in all normal and tumor cell types examined. Tumor cells with a high HspBP1/Hsp70 molar ratio were more susceptible to anticancer drugs than were those with a low ratio. Ectopic expression of HspBP1 enhanced this effect of anticancer drugs in a manner that was both dependent on the ability of HspBP1 to bind to Hsp70 and sensitive to the induction of Hsp70 by mild heat shock. Furthermore, anticancer drugs upregulated HspBP1 expression, whereas prevention of such upregulation by RNA interference reduced the susceptibility of tumor cells to anticancer drugs. Overexpression of HspBP1 promoted the permeabilization of lysosomal membranes, the release of cathepsins from lysosomes into the cytosol, and the activation of caspase-3 induced by anticancer drugs. These results suggest that HspBP1, by antagonizing the prosurvival activity of Hsp70, sensitizes tumor cells to cathepsin-mediated cell death.Members of the 70-kDa heat shock protein (Hsp70) 2 family play an essential role in quality control of cellular proteins (1-3) and include stress-inducible Hsp70, constitutively expressed Hsc70, mitochondrial Hsp75, and endoplasmic reticulum GRP78 (4, 5). Under normal conditions, Hsp70 proteins function as ATP-dependent molecular chaperones by facilitating the folding of newly synthesized polypeptides, the assembly of multiprotein complexes, and the transport of proteins across cellular membranes. Under stressful conditions, the synthesis of inducible Hsp70 enhances the ability of cells to cope with increased concentrations of unfolded or denatured proteins (1-6).The Hsp70 proteins undergo a cycle of substrate binding and release that is accelerated by ATP hydrolysis (1-3). The substrate binding domain of Hsp70 is localized to a 25-kDa COOH-terminal region, with substrate access to this domain being controlled by a COOH-terminal "lid" that exposes the domain in the ATP-bound form and allows substrate binding to occur when Hsp70 is in the ADP-bound form. Opening and closing of the lid are governed by conformational changes associated with ATP binding and hydrolysis, which occur within the cleft of the 45-kDa NH 2 -terminal ATPase domain. The exchange of bound ADP for ATP results in substrate release, thus allowing Hsp70 to enter a new round of substrate binding and release.The chaperone activity of Hsp70 proteins is regulated by various accessory proteins, known as cochaperones (1-3). For example, Hsp40 binds to the COOH terminus of Hsp70, stimulates its AT...
Although the extracellular signal-regulated kinase (ERK) pathway functions downstream of Ras in induction of the cell motility response, the detailed molecular mechanism by which this pathway regulates cell motility has remained elusive. The application of a functional expression cloning strategy to discover proteins that regulate cell motility has resulted in the identification of an SH3 domain-containing protein, SH3P2. Overexpression of SH3P2 in HeLa S3 cells inhibited cell motility, whereas RNA interference-mediated depletion of SH3P2 enhanced motility in various tumor cell lines, suggesting that SH3P2 functions as a negative regulator of cell motility. The expression level of SH3P2 alone did not correlate well with the motility of tumor cells, however. SH3P2 was phosphorylated on Ser 202 by ribosomal S6 kinase (RSK) in an ERK pathway-dependent manner, and such phosphorylation inhibited the ability of SH3P2 to suppress cell motility. The RSK inhibitor BI-D1870 suppressed SH3P2 phosphorylation and tumor cell motility as effectively as did the MEK inhibitor PD184352. Furthermore, expression of the unphosphorylatable SH3P2 mutant SH3P2(S202A) inhibited tumor cell motility, indicating that phosphorylation of SH3P2 at Ser 202 is a key determinant of such motility. These results suggest that SH3P2 is an essential molecule that functions downstream of the ERK pathway to modulate cell motility.
The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.
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