Abstract
ABSTRACTPaxillin is a focal adhesion adaptor protein, heavily phosphorylated at multiple tyrosine residues, as well as at serine 273 (S273), and is known to be critical for cytoskeleton rearrangement and cell migration. We previously found that paxillin plays a regulatory role in IL-3-dependent survival of Ba/F3 cells, a mouse pro-B cell line. In this study, by using overexpressed His6 tagged-paxillin as a bait, we found that DDX42, a DEAD-box RNA helicase, interacted with paxillin, inhibited apoptosis, and promoted polarization of Ba/F3 cells. His6 tagged-paxillin was stably overexpressed in Ba/F3 cells, pulled-down from cell lysates with Ni+-NTA beads, and analyzed by one-dimensional SDS-PAGE followed by LC–MS. We found that DDX42 co-precipitated with paxillin, as demonstrated by western blotting analysis of His6 tagged-paxillin precipitates with anti-DDX42 antibodies and His6 tagged-DDX42 precipitates with anti-paxillin antibodies. In addition, we observed a preferential interaction of DDX42 with the paxillin mutant, S273A, compared to the S273D mutant. Furthermore, DDX42 overexpression in Ba/F3 cells delayed the apoptosis induced by IL-3 deprivation and promoted restoration of the elongated shape in Ba/F3 cells induced by IL-3 re-supply after a 6 h-deprivation. These results suggested that DDX42 interacts with paxillin and participates in IL-3-dependent cell survival, as well as in the cytoskeletal rearrangements underlying polarization of Ba/F3 cells.
Highlights
The interaction of tissue cells with the extracellular matrix is critical for several cellular processes such as differentiation, migration, survival, and growth as well as for clinical conditions such as cancer metastasis (Shan & Zhang 2017; Ojalill et al 2018)
By using overexpressed His6 tagged-paxillin as a bait, we found that DDX42, a DEAD-box RNA helicase, interacted with paxillin, inhibited apoptosis, and promoted polarization of Ba/F3 cells
His6 tagged-paxillin was overexpressed in Ba/F3 cells (Figure 1(A)), pulled-down from the cell lysate with Ni+-NTA beads, washed with 2 M urea, and eluted with 150 mM imidazole, and the co-precipitated proteins were analyzed by one-dimensional SDS-PAGE (Figure 1(B)) followed by LC–MS (Table 1)
Summary
The interaction of tissue cells with the extracellular matrix is critical for several cellular processes such as differentiation, migration, survival, and growth as well as for clinical conditions such as cancer metastasis (Shan & Zhang 2017; Ojalill et al 2018). When cells are attached to the extracellular matrix, a protein complex, the focal adhesion, composed of a high number of signaling and structural proteins, is formed around the cytoplasmic tails of the clustered integrins. Focal adhesion plays a structural role connecting the actin stress fibers to the integrin receptors and transduces bidirectional signals, inside-out and outside-in, contributing to the abovementioned physiological processes (Shen et al 2012). The interaction with actopaxin, in particular, seems to be critical for paxillin connection to the actin stress fibers and for the regulation of actin rearrangements. LIM domains participate in the interaction with PTP-PEST (Jamieson et al 2005), regulating the balance between assembly and disassembly of focal adhesions.
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