LL5beta is a phosphatidylinositol (3,4,5)-trisphosphate sensor that can bind the cytoskeletal adaptor, gamma-filamin.
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 (LL5beta). The protein bound PtdIns(3,4,5)P(3) selectively in vitro. Strikingly, a substantial proportion of LL5beta 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 LL5beta to this vesicle population. Importantly, pleckstrin homology domain mutants of LL5beta 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, LL5beta 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 LL5beta 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, LL5beta bound to the cytoskeletal adaptor, gamma-filamin, tightly and in a PI3K-independent fashion, both in vitro and in vivo. This interaction could co-localize heterologously expressed gamma-filamin with GFP-LL5beta in the unidentified vesicles.
Paranavitane
Varuni
V
Inositide Laboratory, The Babraham Institute, Cambridge CB2 4AT, United Kingdom.
Coadwell
W John
WJ
Eguinoa
Alicia
A
Hawkins
Phillip T
PT
Stephens
Len
L
D000595:Amino Acid Sequence; D001798:Blood Proteins; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D003001:Cloning, Molecular; D003285:Contractile Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010750:Phosphoproteins
D001798:Blood Proteins; D002352:Carrier Proteins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D064448:Filamins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; C464562:FILIP1 protein, human; C471180:PHLDB2 protein, human; C084540:Phldb1 protein, rat; C060974:phosphatidylinositol 3,4,5-triphosphate; C039077:platelet protein P47