Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

Abstract

BackgroundMacropinocytosis, which is a constitutive cellular process of fluid and macromolecule uptake, is regulated by actin cytoskeleton rearrangements near the plasma membrane. Activation of Rac1, which is proposed to act upstream of the actin polymerization regulatory Wave 2 complex, has been found to correlate with enhanced macropinocytosis. One of the components of the Wave 2 complex is Abi1. Multiple, alternatively spliced isoforms of Abi1 are expressed in mammalian cells, but the functional significance of the various isoforms is unknown.Principal FindingsHere, using flow cytometric assay analysis for Alexa Fluor 647, we demonstrate that Abi1 isoforms 2 and 3 differentially regulate macropinocytosis. LNCaP cells expressing isoform 3 had increased macropinocytic uptake that correlated with enhanced cell spreading and higher Rac1 activation in comparison to cells expressing isoform 2. Isoform 2 expressing cells had decreased macropinocytic uptake, but demonstrated greater sensitivity to Rac1 activation. Moreover, more isoform 2 was localized within the cytoplasm in comparison to isoform 3, which was more associated with the plasma membrane. Activated Rac1 was found to specifically bind to a site in exon 10 of isoform 2 in vitro. Because of alternative mRNA splicing, exon 10 is absent from isoform 3, precluding similar binding of activated Rac1. Both isoforms, however, bound to inactive Rac1 through the same non-exon 10 site. Thus, Abi1 isoform 3-containing Wave 2 complex exhibited a differential binding to activated vs. inactive Rac1, whereas isoform 2-containing Wave 2 complex bound activated or inactive Rac1 comparably.ConclusionBased on these observations, we postulate that Abi1 isoforms differentially regulate macropinocytosis as a consequence of their different relative affinities for activated Rac1 in Wave 2 complex. These findings also raise the possibility that isoform-specific roles occur in other Abi1 functions.

miR-199a-3p displays tumor suppressor functions in papillary thyroid carcinoma

“…It is conceivable that the observed lethal phenotype is due to miR-199a-3p action on multiple targets and is mediated by networks including MET, mTOR and molecules involved in macropinocytosis hyperstimulation, as suggested by functional analyses and IPA tool. Among miR-199a-3p targets predicted by integrated analysis, there are indeed genes involved in macropinocytosis, such as ABI1 [52], and other genes worthy of further investigation due to their oncogenic relevance in PTC (listed in supplementary informations). These include C8orf4, also known as thyroid cancer 1 (TC-1) [53], DUSP5, involved in ERK1/2 pathway attenuation [54], and ARG2, whose over-expression has been associated with the thyroid cancer pathogenesis [55].…”

Abi1/Hssh3bp1 pY213 links Abl kinase signaling to p85 regulatory subunit of PI‐3 kinase in regulation of macropinocytosis in LNCaP cells

“…Assay of cellular uptake of Alexa Fluor 647 was performed essentially as described [9], but was quantified by flow cytometry [12]. For uptake assays, following seeding into 12‐well for 24 h and washing, cells were preincubated with 0.2 μM wortmannin or 2 μM imatinib mesylate (STI‐571) (30 min), and incubated in the same medium with or without 10 ng/ml PDGF for 2 h along with Alexa Fluor 647 hydrazide (25 μM).…”

Crk and Abi1: Binary Molecular Switches That Regulate Abl Tyrosine Kinase and Signaling to the Cytoskeleton

“…Moreover, Km allows the comparison of different substrates for the same enzyme. Km of purified GST Abi1 isoform 2 92 was determined using recombinant c-Abl (47aa-end) purified from baculovirus culture treated with Gleevec (STI-571, Novartis Pharmaceuticals, East Hanover, NJ) as described 53 and using reaction conditions as described by Tanis et al 94 The Km of GST Abi1 was 0.33 ± 0.5 µM, and the Km of GST Crk was 0.32 ± 0.02 µM (see below); hence, they are comparable. (B) Abi1 competes with Crk at its Abl SH3 domain binding site.…”