Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

Cyclic AMP stimulates translocation of Na+/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

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Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation

Biochemical Journal ◽

10.1042/bj3150709 ◽

1996 ◽

Vol 315 (3) ◽

pp. 709-713 ◽

Cited By ~ 205

Author(s):

Stephen R. JAMES ◽

C. Peter DOWNES ◽

Roy GIGG ◽

Simon J. A. GROVE ◽

Andrew B. HOLMES ◽

...

Keyword(s):

Plasma Membrane ◽

Surface Plasmon Resonance ◽

Protein Kinase ◽

Association Constant ◽

Kinase Activity ◽

Specific Binding ◽

Protein Kinase Activity ◽

L6 Myotubes ◽

Subsequent Activation ◽

Phosphoinositide 3 Kinase

Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

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The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization

Plant Molecular Biology ◽

10.1007/s11103-013-0061-0 ◽

2013 ◽

Vol 82 (3) ◽

pp. 267-278 ◽

Cited By ~ 35

Author(s):

Sheen X. Lu ◽

Estelle M. Hrabak

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Amino Terminus ◽

Membrane Localization ◽

Dependent Protein Kinase ◽

Calcium Dependent ◽

Plasma Membrane Localization ◽

Dependent Protein ◽

Calcium Dependent Protein Kinase

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α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels

eLife ◽

10.7554/elife.61951 ◽

2021 ◽

Vol 10 ◽

Author(s):

Reeba Susan Jacob ◽

Cedric Eichmann ◽

Alessandro Dema ◽

Davide Mercadante ◽

Philipp Selenko

Keyword(s):

Plasma Membrane ◽

Membrane Fusion ◽

Cellular Function ◽

Helical Conformation ◽

Membrane Localization ◽

Plasma Membrane Localization ◽

Disease Protein ◽

Phosphoinositide 3 Kinase ◽

Acidic Components

The Parkinson's disease protein α-synuclein (aSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate plasma membrane localization of endogenous pools of αSyn in A2780, HeLa, SK-MEL-2 and differentiated and undifferentiated neuronal SH-SY5Y cells. We demonstrate that αSyn binds to reconstituted PIP2-membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute αSyn in cells. We further delineate that αSyn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to αSyn's cellular function(s) at the plasma membrane.

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α-Synuclein plasma membrane localization correlates with cellular phosphatidylinositol polyphosphate levels

10.1101/2020.07.31.224600 ◽

2020 ◽

Author(s):

Cédric Eichmann ◽

Reeba Susan Jacob ◽

Alessandro Dema ◽

Davide Mercadante ◽

Philipp Selenko

Keyword(s):

Plasma Membrane ◽

Membrane Fusion ◽

Cellular Function ◽

Helical Conformation ◽

Membrane Localization ◽

Plasma Membrane Localization ◽

Disease Protein ◽

Phosphoinositide 3 Kinase ◽

Acidic Components

AbstractThe Parkinson’s disease protein α-synuclein (αSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate plasma membrane localization of endogenous pools of αSyn in A2780, HeLa, SH-SY5Y and SK-MEL-2 cells. We demonstrate that αSyn binds reconstituted PIP2-membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute αSyn in cells. We further delineate that αSyn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to αSyn’s cellular function(s) at the plasma membrane.

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Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

Molecular and Cellular Biology ◽

10.1128/mcb.22.17.6247-6260.2002 ◽

2002 ◽

Vol 22 (17) ◽

pp. 6247-6260 ◽

Cited By ~ 236

Author(s):

Michael P. Scheid ◽

Paola A. Marignani ◽

James R. Woodgett

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Protein Kinase B ◽

Membrane Localization ◽

Activation Process ◽

Membrane Recruitment ◽

Molecular Events ◽

Primary Determinant ◽

Serine Kinases ◽

Phosphoinositide 3 Kinase

ABSTRACT The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3′ phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.

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