The Homolog of the Five SH3-Domain Protein (HOFI/SH3PXD2B) Regulates Lamellipodia Formation and Cell Spreading

Abstract

Motility of normal and transformed cells within and across tissues requires specialized subcellular structures, e.g. membrane ruffles, lamellipodia and podosomes, which are generated by dynamic rearrangements of the actin cytoskeleton. Because the formation of these sub-cellular structures is complex and relatively poorly understood, we evaluated the role of the adapter protein SH3PXD2B [HOFI, fad49, Tks4], which plays a role in the development of the eye, skeleton and adipose tissue. Surprisingly, we find that SH3PXD2B is requisite for the development of EGF-induced membrane ruffles and lamellipodia, as well as for efficient cellular attachment and spreading of HeLa cells. Furthermore, SH3PXD2B is present in a complex with the non-receptor protein tyrosine kinase Src, phosphorylated by Src, which is consistent with SH3PXD2B accumulating in Src-induced podosomes. Furthermore, SH3PXD2B closely follows the subcellular relocalization of cortactin to Src-induced podosomes, EGF-induced membrane ruffles and lamellipodia. Because SH3PXD2B also forms a complex with the C-terminal region of cortactin, we propose that SH3PXD2B is a scaffold protein that plays a key role in regulating the actin cytoskeleton via Src and cortactin.

Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

“…4b demonstrates that whereas the wild-type PX domain could bind to a selection of different phosphoinositides, the R43W mutant was practically unable to recognize any of those lipids. We have to note that a weak interaction of the wild-type PX domain was detected with phosphatidylserine, which was not seen in our previous work (19). Finally, we analyzed the intracellular localization of mutant Tks4 proteins.…”

“…Alexa Fluor 488 goat anti-rabbit (A11008) and Alexa Fluor 488 rabbit antimouse (A11059) antibodies were purchased from Invitrogen. Generation of polyclonal anti-Tks4 antibody was described earlier (19). The V5 epitope-tagged Tks4 and PX domain of Tks4 expressed as GST fusion protein were described previously (19).…”

“…Generation of polyclonal anti-Tks4 antibody was described earlier (19). The V5 epitope-tagged Tks4 and PX domain of Tks4 expressed as GST fusion protein were described previously (19). V5-Tks4 Y25F,Y373F,Y508F , V5-Tks4 R71L,R94L , V5-Tks4 R43Q , V5-Tks4 R43W , as well as GST-PX R43W mutants were generated using the QuikChange Site-directed Mutagenesis Kit (Stratagene).…”

“…It has been well established that the PX domain is required for lipid binding (24 -26). To test directly whether the R43W mutation functionally inactivates the PX domain, both wild-type and R43W PX domains were expressed as GST fusion proteins and examined in a protein-lipid overlay assay, as described earlier (19). Fig.…”

“…Recently, we have shown that Tks4 is instrumental for the development of EGF-induced membrane ruffles and lamellipodia as well as for efficient cellular attachment and spreading of HeLa cells (19). In addition, it has been demonstrated that Tks4 associates with Src tyrosine kinase and cortactin, a well established activator of the Arp2/3 complex (19).…”