LL5β Is a Phosphatidylinositol (3,4,5)-Trisphosphate Sensor That Can Bind the Cytoskeletal Adaptor, γ-Filamin

Abstract

We identified a potential phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) binding pleckstrin homology domain in the data bases and have cloned and expressed its full coding sequence (LL5␤). The protein bound PtdIns(3,4,5)P 3 selectively in vitro. Strikingly, a substantial proportion of LL5␤ became associated with an unidentified intracellular vesicle population in the context of low PtdIns(3,4,5)P 3 levels produced by the addition of wortmannin or LY294002. In addition, expression of platelet-derived growth factor-receptor mutants unable to activate type 1A phosphoinositide 3-kinase (PI3K) or serum starvation in porcine aortic endothelial cells lead to redistribution of LL5␤ to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5␤ that could not bind PtdIns(3,4,5)P 3 were constitutively localized to this vesicle population. At increased PtdIns(3,4,5)P 3 levels, LL5␤ was redirected to a predominantly cytoplasmic distribution, presumably through a PI3K-dependent block on its targeting to the vesicular compartment. Furthermore, at high, hormone-stimulated PtdIns-(3,4,5)P 3 levels, it became significantly plasma-membrane localized. The distribution of LL5␤ is thus dramatically and uniquely sensitive to low levels of PtdIns(3,4,5)P 3 indicating it can act as a sensor of both low and hormone-stimulated levels of PtdIns(3,4,5)P 3 . In addition, LL5␤ bound to the cytoskeletal adaptor, ␥-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed ␥-filamin with GFP-LL5␤ in the unidentified vesicles.Phosphoinositide 3-kinases (PI3Ks) 1 3-phosphorylate phosphoinositides. There are three classes of PI3Ks. The type I enzymes seem to act as PtdIns(4,5)P 2 3-kinases in vivo; they can be activated by a variety of close-to-receptor transduction events and drive accumulation of PtdIns(3,4,5)P 3 in the inner leaflet of the plasma membrane. This PtdIns(3,4,5)P 3 serves as signal recruiting proteins from the cytosol that possess modules, typically PH domains, capable of binding its head group (1).There are a variety of reagents that can be used to inhibit PI3K activity. Most widely used are wortmannin (2) and LY294002 (3); both of which potently inhibit nearly all classes of PI3K and hence cannot generally be used to implicate a particular PI3K in a process. More specific are receptor tyrosine kinase Tyr 3 Phe mutants. A number of receptor tyrosine kinases (relevant here, the PDGF ␤-receptor) are capable of binding type IA PI3Ks at specific tyrosine residues that become phosphorylated following ligand binding (4). Mutation of these tyrosines to phenylalanine blocks type IA PI3K binding and activation but does not affect association of other effectors (1, 5). Stable, clonal cell lines have been created overexpressing wild-type PDGF-␤ receptors or (Y740F/Y751F) PDGF-␤ receptors (6) that have allowed the impact of selectivity blocking type IA PI3K activation to be assessed in vivo (7).There is now a substantial famil...

Liprin-α1, ERC1 and LL5 identify a polarized, dynamic compartment implicated in cell migration

“…ERC1a interacts with LL5b (encoded by PHLDB2), a member of the LL5 family of adaptor proteins with a pleckstrin homology (PH) domain, which also includes LL5a (encoded by PHLDB1) and different splice variants. LL5b is part of a stable cortical platform at the periphery of the cell (Paranavitane et al, 2003;Lansbergen et al, 2006;Hotta et al, 2010;Takabayashi et al, 2010). Here, we found that ERC1a and LL5 proteins are needed, together with liprin-a1, to stabilize the edge of migrating cells and for the internalization of active integrins.…”

“…To further dissect the role of liprin-a1 in cell motility, we have looked for relevant liprin-a1-interacting partners. Here, we have identified the adaptor proteins ERC1 (ELKS/Rab6-interacting/CAST family member 1) (Hida and Ohtsuka, 2010;Südhof, 2012) and LL5 (Paranavitane et al, 2003;Kishi et al, 2005) as important co-players, with liprin-a1, in the regulation of tumor cell migration and invasion. The ERC family (Nakata et al, 1999;Monier et al, 2002;Deguchi-Tawarada et al, 2004) includes ERC2, and two ERC1 isoforms, the brainspecific ERC1b and the widely expressed ERC1a (ELKSe) (Wang et al, 2002).…”

Head and neck squamous cell carcinoma transcriptome analysis by comprehensive validated differential display

“…TMEM16A is predicted to be associated with the cell membrane. PHLDB2 is a 160 kDa protein that has, in particular, a C-terminal Pleckstrin homology domain that binds phosphatidylinositol (3,4,5)-triphosphates (Dowler et al, 2000;Paranavitane et al, 2003). The overexpressed sequence we detected lacks exon 25 (data not shown).…”

“…PHLDB2 codes for a 160 kDa protein with a unique spectrin repeat and a C-terminal Pleckstrin homology domain (Paranavitane et al, 2003). Using RT-QPCR, we found that PHLDB2 is overexpressed in 10/12 HNSCC (six-fold on average, Figure 3b).…”

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

“…It is tempting to speculate that by concentrating KANK1 at the FA rims, talin1 helps to 'nucleate' CMSC assembly, which can then propagate to form large structures surrounding FAs ( Figure 6B). Additional membrane-bound cues, such as the presence of PIP3, to which LL5b can bind (Paranavitane et al, 2003), can further promote CMSC coalescence by increasing concentration of CMSC players in specific areas of the plasma membrane. This model helps to explain why the CMSC accumulation at the cell periphery is reduced but not abolished when PI3 kinase is inhibited (Lansbergen et al, 2006), and why the clustering of all CMSC components is mutually dependent.…”