scholarly journals Phosphatidylinositol-4-Kinase Type II Alpha Contains an AP-3–sorting Motif and a Kinase Domain That Are Both Required for Endosome Traffic

2008 ◽  
Vol 19 (4) ◽  
pp. 1415-1426 ◽  
Author(s):  
Branch Craige ◽  
Gloria Salazar ◽  
Victor Faundez
Keyword(s):  
Synaptic Vesicles ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Membrane Domains ◽  
Type Ii ◽  
Catalytic Domains ◽  
Sorting Motif ◽  
Lysosome Related Organelles ◽  
Mutant Phenotypes ◽  
Phosphatidylinositol 4

The adaptor complex 3 (AP-3) targets membrane proteins from endosomes to lysosomes, lysosome-related organelles and synaptic vesicles. Phosphatidylinositol-4-kinase type II α (PI4KIIα) is one of several proteins possessing catalytic domains that regulate AP-3–dependent sorting. Here we present evidence that PI4KIIα uniquely behaves both as a membrane protein cargo as well as an enzymatic regulator of adaptor function. In fact, AP-3 and PI4KIIα form a complex that requires a dileucine-sorting motif present in PI4KIIα. Mutagenesis of either the PI4KIIα-sorting motif or its kinase-active site indicates that both are necessary to interact with AP-3 and properly localize PI4KIIα to LAMP-1–positive endosomes. Similarly, both the kinase activity and the sorting signal present in PI4KIIα are necessary to rescue endosomal PI4KIIα siRNA-induced mutant phenotypes. We propose a mechanism whereby adaptors use canonical sorting motifs to selectively recruit a regulatory enzymatic activity to restricted membrane domains.

scholarly journals Phosphatidylinositol 4-kinase type II  is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles

2003 ◽  
Vol 100 (7) ◽  
pp. 3995-4000 ◽  
Author(s):  
J. Guo ◽  
M. R. Wenk ◽  
L. Pellegrini ◽  
F. Onofri ◽  
F. Benfenati ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Kinase Activity ◽  
Type Ii ◽  
Phosphatidylinositol 4

Nuclear localization of phosphatidylinositol 4-kinase β

2002 ◽  
Vol 115 (8) ◽  
pp. 1769-1775 ◽  
Author(s):  
Petra de Graaf ◽  
Elsa E. Klapisz ◽  
Thomas K. F. Schulz ◽  
Alfons F. M. Cremers ◽  
Arie J. Verkleij ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Kinase Activity ◽  
Insoluble Fraction ◽  
Type Ii ◽  
3T3 Cells ◽  
Type Iii ◽  
Pore Complexes ◽  
Phosphatidylinositol 4 ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Whereas most phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity is localized in the cytoplasm, PtdIns 4-kinase activity has also been detected in membranedepleted nuclei of rat liver and mouse NIH 3T3 cells. Here we have characterized the PtdIns 4-kinase that is present in nuclei from NIH 3T3 cells. Both type II and type III PtdIns 4-kinase activity were observed in the detergent-insoluble fraction of NIH 3T3 cells. Dissection of this fraction into cytoplasmic actin filaments and nuclear lamina-pore complexes revealed that the actin filament fraction contains solely type II PtdIns 4-kinase,whereas lamina-pore complexes contain type III PtdIns 4-kinase activity. Using specific antibodies, the nuclear PtdIns 4-kinase was identified as PtdIns 4-kinase β. Inhibition of nuclear export by leptomycin B resulted in an accumulation of PtdIns 4-kinase β in the nucleus. These data demonstrate that PtdIns 4-kinase β is present in the nuclei of NIH 3T3 fibroblasts,suggesting a specific function for this kinase in nuclear processes.

scholarly journals Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

2020 ◽  
Author(s):  
Sven H. Schmidt ◽  
Jui-Hung Weng ◽  
Phillip C. Aoto ◽  
Daniela Boassa ◽  
Sebastian Mathea ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Conformational Dynamics ◽  
Subcellular Location ◽  
Kinase Domain ◽  
Activation Process ◽  
Type I ◽  
Rab Gtpases ◽  
Exchange Mass Spectrometry ◽  
Catalytic Domains ◽  
Dynamics Simulations

AbstractIn a multi-tiered approach, we explored how Parkinson’s Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYGψ motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYGψ motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

Molecular determinants of activation and membrane targeting of phosphoinositol 4-kinase IIβ

2007 ◽  
Vol 409 (2) ◽  
pp. 501-509 ◽  
Author(s):  
Gwanghyun Jung ◽  
Jing Wang ◽  
Pawel Wlodarski ◽  
Barbara Barylko ◽  
Derk D. Binns ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Type Ii ◽  
Lipid Kinase ◽  
Catalytic Domains ◽  
Specific Behaviour ◽  
Molecular Determinants

Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIα and β. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1–90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIα behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIβ, only 50% of PI4KIIβ is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIβ to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIβ undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIβ is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIβ is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and α/β chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIα and β.

Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1

2002 ◽  
Vol 363 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Christina PANARETOU ◽  
Sharon A. TOOZE
Keyword(s):  
Kinase Activity ◽  
Secretory Granules ◽  
Specific Inhibitor ◽  
Small Gtpases ◽  
Type Ii ◽  
Adp Ribosylation ◽  
Lipid Kinases ◽  
Phosphatidylinositol 4 ◽  
Golgi Network ◽  
Adp Ribosylation Factor

Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive Gi/G0 proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913–16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on Gi/G0 activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1—GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280–287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962–13170], whereas ARF1—GTP does regulate the production of PtdIns(4,5)P2. Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.

Signalling and non-caveolar rafts

2001 ◽  
Vol 29 (4) ◽  
pp. 509-512 ◽  
Author(s):  
M. G. Waugh ◽  
S. Minogue ◽  
J. S. Anderson ◽  
M. dos Santos ◽  
J. J. Hsuan
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Phosphatidyl Inositol ◽  
Carcinoma Cells ◽  
Density Gradients ◽  
Membrane Domains ◽  
Type Ii ◽  
Epidermal Growth ◽  
Phosphatidylinositol 4

Rafts are small membrane domains containing discrete subsets of lipids and proteins. Although microscopic raft structures termed ‘caveolae’ were described nearly 50 years ago, the importance of rafts, particularly signalling within rafts, is only beginning to be understood. Our studies focus on receptor-dependent phosphoinositide signalling. Using their characteristic buoyancy in density gradients, we and others found that the epidermal growth factor (EGF) receptor, phosphatidyl-inositol 4-kinase and phosphoinositides are localized within a caveolin-rich fraction of A431 carcinoma cells. We subsequently found that membrane fragments containing the EGF receptor and most cellular phosphoinositides can be separated from caveolae. Consequently, components of EGF-dependent phosphoinositide signalling localize to one or more novel types of raft, the composition of which we are currently determining. A key component is the type II phosphatidylinositol 4-kinase, which, for many years, has proven difficult to purify and clone. We describe our recent purification from rafts and cloning of this elusive enzyme, and discuss how the structure sheds light on the rafting of this enzyme.

scholarly journals BLOC-1 Complex Deficiency Alters the Targeting of Adaptor Protein Complex-3 Cargoes

2006 ◽  
Vol 17 (9) ◽  
pp. 4014-4026 ◽  
Author(s):  
G. Salazar ◽  
B. Craige ◽  
M. L. Styers ◽  
K. A. Newell-Litwa ◽  
M. M. Doucette ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Complexes ◽  
Adaptor Protein ◽  
Cellular Mechanism ◽  
Type Ii ◽  
Mouse Mutants ◽  
Late Endosomes ◽  
Complex Deficiency ◽  
Lysosome Related Organelles ◽  
Phosphatidylinositol 4

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1–deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.

scholarly journals An early endosome–derived retrograde trafficking pathway promotes secretory granule maturation

2020 ◽  
Vol 219 (3) ◽  
Author(s):  
Cheng-I J. Ma ◽  
Yitong Yang ◽  
Taeah Kim ◽  
Chang Hua Chen ◽  
Gordon Polevoy ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Biologically Active ◽  
Type Ii ◽  
Endosomal Sorting ◽  
Trafficking Pathway ◽  
Granule Maturation ◽  
Lysosome Related Organelles ◽  
Phosphatidylinositol 4 ◽  
Endosomal System ◽  
External Stimuli

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi–derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.

scholarly journals Conformation and Dynamics of the Kinase Domain Drive Subcellular Location and Activation of LRRK2.

2020 ◽  
Author(s):  
Sven H. Schmidt ◽  
Jui-Hung Weng ◽  
Phillip C. Aoto ◽  
Daniela Boassa ◽  
Steven Silletti ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Full Length ◽  
Filament Formation ◽  
Catalytic Domains ◽  
Dynamics Simulations ◽  
Hdx Ms

Abstract Background: Leucine-Rich Repeat Kinase 2 (LRRK2) is a complex multi-domain protein where LRRK2-mutations are associated with Parkinson´s Disease (PD). To explore how pathogenic PD-mutations hijack the finely tuned activation process of LRRK2, we here used a multi-tiered approach. Methods: First, the spatial and temporal distribution of full-length LRRK2 was investigated by a real-time cell-based assay in the presence and absence of LRRK2-kinase inhibitors. In a 2nd layer we explored the consequences of PD mutations as well as removal of the N-terminal domains employing a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We focused on the biochemical characterization of LRRK2RCKW variants based on kinase assays using Rab8a or LRRKtide as substrates. Next, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the solvent accessible regions of LRRK2RCKW in the presence and absence of the LRRK2 inhibitor MLi-2. Finally, Molecular Dynamics simulations on the kinase domain were applied to elucidate differences in breathing dynamics between wild type and mutants of the DYGψ motif. Results: Our cellular approaches revealed that the kinase inhibitors MLi-2 and rebastinib both freeze the kinase domain in a stable conformation, however, only MLi-2 resembles an active conformation and induces filament formation. LRRK2RCKW showed, regardless of the mutation it was combined with, filament formation, indicating a shielding function of the N-terminal domains. This shielding function is impaired for pathogenic mutations in full length LRRK2. LRRK2RCKW retained kinase activity similar to full-length LRRK2. HDX-MS provided a comprehensive allosteric portrait of the kinase domain and revealed how MLi-2 binding is sensed by the entire protein. Molecular Dynamics simulations suggest that, while all domains contribute to regulating kinase activity and spatial distribution, it is the highly dynamic kinase domain, driven by the DYGψ motif, that coordinates the overall domain crosstalk and serves as a regulatory hub for the intrinsic regulation of LRRK2.Conclusion: These studies confirm our hypothesis that the N-terminal scaffolding domains shield the catalytic domains in an inactive state. PD mutations, MLi-2, or Rab GTPases can all unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules.

scholarly journals Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes

1992 ◽  
Vol 284 (1) ◽  
pp. 39-45 ◽  
Author(s):  
A Graziani ◽  
L E Ling ◽  
G Endemann ◽  
C L Carpenter ◽  
L C Cantley
Keyword(s):  
Monoclonal Antibody ◽  
Human Erythrocyte ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Bovine Brain ◽  
Erythrocyte Membranes ◽  
Major Protein ◽  
Type Ii ◽  
Effective Inhibitor ◽  
Phosphatidylinositol 4

PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.

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