Accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in thrombin-stimulated platelets. Different sensitivities to Ca2+ or functional integrin.
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ABSTRACT:
SUBMITTER: Sorisky A
PROVIDER: S-EPMC1132936 | biostudies-other | 1992 Sep
REPOSITORIES: biostudies-other
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Project description:Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P(3) regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P(3) and the reciprocal regulation of PtdIns(3,4,5)P(3) itself. LL5beta, whose subcellular localization is directed by membrane PtdIns(3,4,5)P(3), recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P(3) accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5beta by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5beta or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5beta without its PtdIns(3,4,5)P(3)-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P(3) accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P(3) at the same location.
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Project description:mTOR serves as a central regulator of cell growth and metabolism by forming two distinct complexes, mTORC1 and mTORC2. Although mechanisms of mTORC1 activation by growth factors and amino acids have been extensively studied, the upstream regulatory mechanisms leading to mTORC2 activation remain largely elusive. Here, we report that the pleckstrin homology (PH) domain of SIN1, an essential and unique component of mTORC2, interacts with the mTOR kinase domain to suppress mTOR activity. More importantly, PtdIns(3,4,5)P3, but not other PtdInsPn species, interacts with SIN1-PH to release its inhibition on the mTOR kinase domain, thereby triggering mTORC2 activation. Mutating critical SIN1 residues that mediate PtdIns(3,4,5)P3 interaction inactivates mTORC2, whereas mTORC2 activity is pathologically increased by patient-derived mutations in the SIN1-PH domain, promoting cell growth and tumor formation. Together, our study unravels a PI3K-dependent mechanism for mTORC2 activation, allowing mTORC2 to activate AKT in a manner that is regulated temporally and spatially by PtdIns(3,4,5)P3.The SIN1-PH domain interacts with the mTOR kinase domain to suppress mTOR activity, and PtdIns(3,4,5)P3 binds the SIN1-PH domain to release its inhibition on the mTOR kinase domain, leading to mTORC2 activation. Cancer patient-derived SIN1-PH domain mutations gain oncogenicity by loss of suppressing mTOR activity as a means to facilitate tumorigenesis.
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Project description:Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.
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Project description:We describe new signalling consequences for PPIP5K1 (diphosphoinositol pentakisphosphate kinase type 1)-mediated phosphorylation of InsP6 and 5-InsP7 to 1-InsP7 and InsP8. In NIH 3T3 cells, either hyperosmotic stress or receptor activation by PDGF (platelet-derived growth factor) promoted translocation of PPIP5K1 from the cytoplasm to the plasma membrane. The PBD1 (polyphosphoinositide-binding domain) in PPIP5K1 recapitulated that translocation. Mutagenesis of PBD1 to reduce affinity for PtdIns(3,4,5)P3 prevented translocation. Using surface plasmon resonance, we found that PBD1 association with vesicular PtdIns(3,4,5)P3 was inhibited by InsP6 and diphosphoinositol polyphosphates. However, the inhibition by PPIP5K1 substrates (IC50: 5-InsP7=5 μM and InsP6=7 μM) was substantially more potent than that of the PPIP5K1 products (IC50: InsP8=32 μM and 1-InsP7=43 μM). This rank order of ligand competition with PtdIns(3,4,5)P3 was also exhibited by the PH (pleckstrin homology) domains of Akt (also known as protein kinase B), GRP1 (general receptor for phosphoinositides 1) and SIN1 (stress-activated protein kinase-interaction protein 1). We propose that, in vivo, PH domain binding of InsP6 and 5-InsP7 suppresses inappropriate signalling ('noise') from stochastic increases in PtdIns(3,4,5)P3. That restraint may be relieved by localized depletion of InsP6 and 5-InsP7 at the plasma membrane following PPIP5K1 recruitment. We tested this hypothesis in insulin-stimulated L6 myoblasts, using mTOR (mechanistic/mammalian target of rapamycin)-mediated phosphorylation of Akt on Ser473 as a readout for SIN1-mediated translocation of mTORC (mTOR complex) 2 to the plasma membrane [Zoncu, Efeyan and Sabatini (2011) Nat. Rev. Mol. Cell Biol. 12, 21-35]. Knockdown of PPIP5K1 expression was associated with a 40% reduction in Ser473 phosphorylation. A common feature of PtdIns(3,4,5)P3-based signalling cascades may be their regulation by PPIP5K1.
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Project description:Prior to ovulation, mammalian oocytes complete their first meiotic division and arrest at metaphase II. During this marked asymmetric cell division, the meiotic spindle moves dramatically from the center of the oocyte to the cortex to facilitate segregation of half of its chromosomal content into the diminutive first polar body. Recent investigations have documented crucial roles for filamentous actin (F-actin) in meiotic spindle translocation. However, the identity of the upstream regulators responsible for these carefully orchestrated movements has remained elusive. Utilizing fluorescently tagged probes and time-lapse confocal microscopy, we document that phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is constitutively synthesized with spatial and temporal dynamics similar to that of F-actin and Formin 2 (Fmn2). Blockage of PtdIns(3,4,5)P3 synthesis by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), disrupts cytoplasmic F-actin organization and meiotic spindle migration to the cortex. F-actin nucleator Fmn2 and Rho GTPase Cdc42 play roles in mediating the effect of PtdIns(3,4,5)P3 on F-actin assembly. Moreover, the spatial and temporal dynamics of PtdIns(3,4,5)P3 is impaired by depletion of MATER or Filia, two oocyte proteins encoded by maternal effect genes. Thus, PtdIns(3,4,5)P3 is synthesized during meiotic maturation and acts upstream of Cdc42 and Fmn2, but downstream of MATER/Filia proteins to regulate the F-actin organization and spindle translocation to the cortex during mouse oocyte meiosis.
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