Abstract
Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830–840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250–1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays an important role in endocytosis.
Highlights
Actin is a highly conserved essential component of the cytoskeleton that generates forces to drive cell motility, cell division, and cell adhesion, maintains cell morphology and polarity, and functions in endocytosis and vesicular trafficking [1]
These results suggest that phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 controls organization of actin filaments through novel mechanisms distinct from other Actin filament-associated protein (AFAP) family members
These results indicated that a C-terminal sequence of approximately 70 amino acids was required for localization to actin filaments. With these results, a deletion mutant consisting of C-terminal 69 amino acid residues [775–843] showed filamentous patterns corresponding to actin filaments (Figure 1B). These results showed that a region responsible for PI3KAP/ XB130 colocalization with actin filaments existed within its C-terminal region 775-843
Summary
Actin is a highly conserved essential component of the cytoskeleton that generates forces to drive cell motility, cell division, and cell adhesion, maintains cell morphology and polarity, and functions in endocytosis and vesicular trafficking [1]. The actin-binding domain (ABD) of AFAP-110 is conserved in AFAP1L1, which has been reported to be localized to the actin-based structure, invadosome [10] In contrast to these two AFAP family members, the ABD is not conserved in PI3KAP/ XB130. It has been reported that PI3KAP/XB130 is localized to cortical actin structures including lamellipodia [16, 17] These results suggest that PI3KAP/XB130 controls organization of actin filaments through novel mechanisms distinct from other AFAP family members
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