Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.     

Common and distinct roles for the binding partners Rabenosyn-5 and Vps45 in the regulation of endocytic trafficking in mammalian cells

In several invertebrate organisms, the Sec1p/Munc18-like protein Vps45 interacts with the divalent Rab4/Rab5 effector, Rabenosyn-5 and carries out multiple functions in the endocytic/secretory pathways. In mammalian cells, Vps45 and Rabenosyn-5 also interact, but the molecular characterization of this binding, and the functional relationship between these two proteins has not been well defined. Here we identify a novel sequence within Rabenosyn-5 required for its interaction with Vps45. We demonstrate that hVps45-depletion decreases expression of Rabenosyn-5, likely resulting from Rabenosyn-5 degradation through the proteasomal pathway. Furthermore, we demonstrate that similar to Rabenosyn-5-depletion, hVps45-depletion causes impaired recycling of β1 integrins, and a subsequent delay in human fibroblast cell migration on fibronectin-coated plates. Moreover, β1 integrin recycling could be rescued by reintroduction of siRNA-resistant wild-type Rabenosyn-5, but not a mutant deficient in Vps45 binding. However, unlike Rabenosyn-5-depletion, which induces Golgi fragmentation and decreased recruitment of sorting nexin retromer subunits to the Golgi, hVps45-depletion induces Golgi condensation and accumulation of retromer subunits in the vicinity of the Golgi. In part, these phenomena could be attributed to reduced Syntaxin16 expression and altered localization of both Syntaxin16 and Syntaxin6 upon Vps45-depletion. Overall, these findings implicate hVps45 and Rabenosyn-5 in post early endosome transport, and we propose that their interaction serves as a nexus to promote bidirectional transport along the endosome-to-recycling compartment and endosome-to-Golgi axes.     

Role of the alpha(1) integrin cytoplasmic tail in the formation of focal complexes, actin organization, and in the control of cell migration

Integrins play a key role in cellular motility; an essential process for embryonic development and tissue morphogenesis, and also for pathological processes such as tumor cell invasion and metastasis. Recently, we showed that the cytoplasmic tail of integrin alpha(1) regulates the formation of focal complexes, F-actin cytoskeleton reorganization, and migration. We now report that the alpha(1) tail directly engages in collagen IV-mediated migration by regulation of the small GTPase Rac1. Deletion variants of the alpha(1) integrin differ in their ability to activate Rac1. Constitutively active Rac1 rescues motility in otherwise immotile cells expressing a truncated alpha(1) integrin without any cytoplasmic tail. In these cells, levels of GTP-Rac1 are constitutively elevated, but kept non-functional in the cytoplasm. The conserved GFFKR motif is sufficient to convey Rac1 activation, but downregulates the amount of GTP-Rac1 in the absence of the alpha(1)-specific sequence PLKKKMEK. This sequence is also required for the recruitment of PI3K to focal adhesions following Rac1 activation. Our results demonstrate that the short alpha(1) cytoplasmic tail is crucial for Rac1 activation and PI3K localization, which in turn results in cytoskeletal rearrangement and subsequent migration.     

Identification and characterization of splicing variants of PLEKHA5 (Plekha5) during brain development

PLEKHA5 (pleckstrin homology domain-containing protein family A, member 5) belongs to the PLEKHA family (PLEKHA1-6); however, the properties of this protein remain poorly characterized. We have identified and characterized two forms of PLEKHA5 mRNA. The long form of PLEKHA5 (L-PLEKHA5) contains 32 exons, encodes 1282 amino acids, and is specifically expressed in the brain; the short form of PLEKHA5 (S-PLEKHA5) is generated by alternative splicing of L-PLEKHA5, contains 26 exons, encodes 1116 amino acids, and is ubiquitously expressed. Both forms of the protein contain putative Trp-Trp (WW) and pleckstrin homology (PH) domains and are located mainly in the cytosol. Developmental and age-dependent expression studies in the mouse brain have shown that Plekha5 is the most abundantly expressed protein at E13.5 with S-Plekha5 dominancy. L-Plekha5 levels increased gradually with the decrease in total Plekha5 levels; moreover, L-Plekha5 became the dominant protein at E17.5, maintaining its dominance throughout adulthood. Protein-lipid overlay assays have indicated that the PH domain of PLEKHA5 specifically interacts with PI3P, PI4P, PI5P, and PI(3,5)P2. These results suggest that the S- to L-conversion of PLEKHA5 (Plekha5) may play an important role in brain development through association with specific phosphoinositides.     

Simultaneous loss of the DLC1 and PTEN tumor suppressors enhances breast cancer cell migration

The phosphatase and tensin homolog (PTEN) gene is a tumor suppressor frequently deleted or mutated in sporadic tumors of the breast, prostate, endometrium and brain. The protein acts as a dual specificity phosphatase for lipids and proteins. PTEN loss confers a growth advantage to cells, protects from apoptosis and favors cell migration. The deleted in liver cancer 1 (DLC1) gene has emerged as a novel tumor suppressor downregulated in a variety of tumor types including those of the breast. DLC1 contains a Rho GTPase activating domain that is involved in the inhibition of cell proliferation, migration and invasion. To investigate how simultaneous loss of PTEN and DLC1 contributes to cell transformation, we downregulated both proteins by RNA interference in the non-invasive MCF7 breast carcinoma cell line. Joint depletion of PTEN and DLC1 resulted in enhanced cell migration in wounding and chemotactic transwell assays. Interestingly, both proteins were found to colocalize at the plasma membrane and interacted physically in biochemical pulldowns and coimmunoprecipitations. We therefore postulate that the concerted local inactivation of signaling pathways downstream of PTEN and DLC1, respectively, is required for the tight control of cell migration.     

Apical membrane segregation of phosphatidylinositol-4,5-bisphosphate influences parathyroid hormone 1 receptor compartmental signaling and localization via direct regulation of ezrin in LLC-PK1 cells

The parathyroid hormone 1 receptor (PTH1R), a primary regulator of mineral ion homeostasis, is expressed on both the apical and basolateral membranes of kidney proximal tubules and in the LLC-PK1 kidney cell line. In LLC-PK1 cells, apical PTH1R subpopulations are far more effective at signaling via phospholipase (PLC) than basolateral counterparts, revealing the presence of compartmental signaling. Apical PTH1R localization is dependent upon direct interactions with ezrin, an actin-membrane cross-linking scaffold protein. Ezrin undergoes an activation process that is dependent upon phosphorylation and binding to phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid that is selectively concentrated to apical surfaces of polarized epithelia. Consistently, the intracellular probe for PIP2, GFP-PLCδ1-PH, localizes to the apical membranes of LLC-PK1 cells, directly overlapping ezrin and PTH1R expression. Activation of the apical PTH1R shifts the GFP-PLCδ1-PH probe from the apical membrane to the cytosol and basolateral membranes, reflecting domain-specific activation of PLC and hydrolysis of PIP2. This compartmental signaling is likely due to the polarized localization of PIP2, the substrate for PLC. PIP2 degradation using a membrane-directed phosphatase shifts ezrin localization to the cytosol and induces ezrin de-phosphorylation, processes consistent with inactivation. PIP2 degradation also shifts PTH1R expression from brush border microvilli to basolateral membranes and markedly blunts PTH-elicited activation of the MAPK pathway. Transient expression of ezrin in HEK293 cells shifts PTH1R expression from the plasma membrane to microvilli-like surface projections that also contain PIP2. As a result, ezrin enhances PTH mediated activation of the PLC pathway in this cell model with increasing total receptor surface expression. Collectively, these findings demonstrate that the apical segregation of PIP2 to the apical domains not only promotes the activation of ezrin and the subsequent formation of the PTH1R containing scaffold, but also ensures the presence of ample substrate for propagating the PLC pathway.     

Requirement of phosphatidylinositol-4,5-bisphosphate for HERC1-mediated guanine nucleotide release from ARF proteins

HERC1 is a giant multidomain protein involved in membrane trafficking through its interaction with vesicle coat proteins such as clathrin and ARF. Previously, it has been shown that the RCC1-like domain 1 (RLD1) of HERC1 stimulates guanine nucleotide dissociation on ARF1 and Rab proteins. In this study, we have analyzed whether HERC1 may also regulate ARF6 activity. We show that HERC1, through its RLD1, stimulates GDP release from ARF6 but, unexpectedly, it inhibits GDP/GTP exchange on ARF6 under conditions where ARNO stimulates it. Furthermore, we demonstrate that the activity of HERC1 as a guanine nucleotide release factor requires the presence of PI(4,5)P(2) bound to HERC1's RLD1. In agreement with this, we find that purified HERC1 contains PI(4,5)P(2) bound to the RLD1.     

The Drosophila DOCK family protein sponge is involved in differentiation of R7 photoreceptor cells

The Drosophila sponge (spg)/CG31048 gene belongs to the dedicator of cytokinesis (DOCK) family genes that are conserved in a wide variety of species. DOCK family members are known as DOCK1–DOCK11 in mammals. Although DOCK1 and DOCK2 involve neurite elongation and immunocyte differentiation, respectively, the functions of other DOCK family members are not fully understood. Spg is a Drosophila homolog of mammalian DOCK3 and DOCK4. Specific knockdown of spg by the GMR-GAL4 driver in eye imaginal discs induced abnormal eye morphology in adults. To mark the photoreceptor cells in eye imaginal discs, we used a set of enhancer trap strains that express lacZ in various sets of photoreceptor cells. Immunostaining with anti-Spg antibodies and anti-lacZ antibodies revealed that Spg is localized mainly in R7 photoreceptor cells. Knockdown of spg by the GMR-GAL4 driver reduced signals of R7 photoreceptor cells, suggesting involvement of Spg in R7 cell differentiation. Furthermore, immunostaining with anti-dpERK antibodies showed the level of activated ERK signal was reduced extensively by knockdown of spg in eye discs, and both the defects in eye morphology and dpERK signals were rescued by over-expression of the Drosophila raf gene, a component of the ERK signaling pathway. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rap1 in and around the plasma membrane of the eye disc cells. Together, these results indicate Spg positively regulates the ERK pathway that is required for R7 photoreceptor cell differentiation and the regulation is mediated by interaction with Rap1 during development of the compound eye.     

Inhibition of SH2-domain containing inositol phosphatase 2 (SHIP2) in insulin producing INS1E cells improves insulin signal transduction and induces proliferation

Inhibition of the lipid phosphatase SH2-domain containing inositol phosphatase 2 (SHIP2) in L6-C10 muscle cells, in 3T3-L1 adipocytes and in the liver of db/db mice has been shown to ameliorate insulin signal transduction and established SHIP2 as a negative regulator of insulin action. Here we show that SHIP2 inhibition in INS1E insulinoma cells increased Akt, glycogen synthase kinase 3 and extracellular signal-regulated kinases 1 and 2 phosphorylation. SHIP2 inhibition did not prevent palmitate-induced apoptosis, but increased cell proliferation. Our data raise the interesting possibility that SHIP2 inhibition exerts proliferative effects in beta-cells and further support the attractiveness of a specific inhibition of SHIP2 for the treatment of type 2 diabetes.     

Regulation of lamellipodia formation and cell invasion by CLIP-170 in invasive human breast cancer cells

Lamellipodia formation necessary for cell invasion is regulated by Rac1. We report here that lamellipodia formation and three-dimensional invasion were significantly promoted by HGF and serum, respectively, in invasive human breast cancer cells. Rac1 formed a complex with CLIP-170, IQGAP1, and kinesin in serum-starved cells, and stimulation of the cells with HGF and serum caused the partial release of IQGAP1 and kinesin from Rac1-CLIP-170 complex. The HGF-induced release of the proteins and promotion of lamellipodia formation were inhibited by an inhibitor of PI3K. Moreover, downregulation of CLIP-170 by siRNA released IQGAP1 and kinesin from Rac1 and promoted lamellipodia formation and invasion, independent of HGF and serum. The results suggest that promotion of lamellipodia formation and invasion by HGF or serum requires PI3K-dependent release of IQGAP1 and kinesin from Rac1-CLIP-170 complex and that CLIP-170 prevents cells from the extracellular stimulus-independent lamellipodia formation and invasion by tethering IQGAP1 and kinesin to Rac1.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.     

Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells

Rab5 is a GTP-binding protein that is crucial for endocytic machinery functions. We previously identified L-plastin as a binding protein for Rab5, using an affinity column with constitutively active Rab5. L- and T-plastin are isoforms of a plastin protein family belonging to actin-bundling proteins that are implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. However, the physiological relevance of Rab5 binding to plastin has remained unclear. Here, we show that L- and T-plastin interacted only with activated Rab5 and that they co-localized with Rab5 on the plasma membrane and endosome. Rab5 activity was also higher in both L- and T-plastin over-expressing Cos-1 cells. Furthermore, expression of L- and T-plastin increased the rate of fluid-phase endocytosis. These findings imply that the Rab5 is either activated or the activity is sustained by interaction with plastin, and that this interaction influences endocytic activity.     

Regulation of phospholipase D by phosphorylation-dependent mechanisms

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4,5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Cα as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.     

Dynamic phospholipid signaling by G protein-coupled receptors

G protein-coupled receptors (GPCRs) control a variety of fundamental cellular processes by regulating phospholipid signaling pathways. Essential for signaling by a large number of receptors is the hydrolysis of the membrane phosphoinositide PIP₂ by phospholipase C (PLC) into the second messengers IP₃ and DAG. Many receptors also stimulate phospholipase D (PLD), leading to the generation of the versatile lipid, phosphatidic acid. Particular PLC and PLD isoforms take differential positions in receptor signaling and are additionally regulated by small GTPases of the Ras, Rho and ARF families. It is now recognized that the PLC substrate, PIP₂, has signaling capacity by itself and can, by direct interaction, affect the activity and subcellular localization of PLD and several other proteins. As expected, the synthesis of PIP₂ by phosphoinositide 5-kinases is tightly regulated as well. In this review, we present an overview of how these signaling pathways are governed by GPCRs, explain the molecular basis for the spatially and temporally organized, highly dynamic quality of phospholipid signaling, and point to the functional connection of the pathways.     

Expression and function of phospholipase C in breast carcinoma

Phosphoinositide-specific phospholipase C (PLC) control the levels of their substrate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), and its hydrolysis products diacylglycerol (DAG) and Ins(1,4,5)P₃, second messengers key to growth control and cell movement. The former is modulated by breakdown of plasma membrane and nuclear phosphoinositides, while the latter is mediated by phosphoinositide-driven remodeling of the actin cytoskeleton. The roles of PLC in the etiology and progression of breast carcinoma, however, are poorly understood. Previous studies reported a correlation between PLCβ₂ expression and breast tumor grade, making PLCβ₂ a potential marker for clinical outcome (Bertagnolo et al., 2006). While over-expression of PLCβ₂ is not sufficient to induce transformation of normal breast epithelial cells, it appears to play a role in promoting cell migration (Bertagnolo et al., 2007).Here we examined the expression of this and other PLC mRNA (β₁–β₄, δ₁, δ₃ and δ₄, γ₁ and γ₂) in normal breast epithelial lines, MCF-10A, and compared that pattern to breast tumor lines MDA-MB-231 and to T47D, using real-time relative-quantification PCR. Our results show that PLCγ₁, γ₂ and δ₁ and δ3 are more highly expressed in the transformed cell lines compared to MCF-10A when normalized to mRNA encoding various house keeping proteins; whereas PLCβ₂ mRNA levels were considerably lower than other PLC subtypes, including PLCβ₁ in the metastatic lines. Examination of PLC mRNA levels from normal and cancerous human breast tissue showed a similar pattern of expression, however, when staging or tumor size was considered, PLCδ₁ and δ₃ expression were positively correlated.To test whether PLCδ₁ or δ₃ played any role in tumor cell proliferation or cell migration, we transfected cells with siRNA specifically targeting these isoforms. RNAi mediated knockdown of either PLC isoform, reduced proliferation of the MDA-MB-231 cells. Morphological changes including cell rounding, and surface blebbing and nuclear fragmentation were observed. These changes were accompanied by reductions in cell migration activities. On the other hand, PLCδ₁ knockdown failed to cause comparable morphological changes in the normal MCF-10A line, but did reduce cell proliferation and migration. Taken together, these data are consistent with the idea that PLCδ₁ and δ₃ isoforms support the growth and migration of normal and neoplastic mammary epithelial cells in vitro.     

Diacylglycerol kinase ζ: at the crossroads of lipid signaling and protein complex organization

Diacylglycerol (DAG) and phosphatidic acid (PA) are lipids with unique functions as metabolic intermediates, basic membrane constituents, and second-signal components. Diacylglycerol kinases (DGK) regulate the levels of these two lipids, catalyzing the interconversion of one to the other. The DGK family of enzymes is composed of 10 isoforms, grouped into five subfamilies based on the presence of distinct regulatory domains. From its initial characterization as a type IV DGK to the generation of mouse models showing its importance in cardiac dysfunction and immune pathologies, diacylglycerol kinase ζ (DGKζ) has proved an excellent example of the critical role of lipid-metabolizing enzymes in the control of cell responses. Although the mechanism that regulates this enzyme is not well known, many studies demonstrate its subtle regulation and its strategic function in specific signaling and as part of adaptor protein complexes. These data suggest that DGKζ offers new opportunities for therapeutic manipulation of lipid metabolism.     

Inhibition of ADP-ribosylation suppresses aberrant accumulation of lipidated apolipoprotein B in the endoplasmic reticulum

ApoB-crescent, an endoplasmic reticulum (ER)-lipid droplet amalgamation structure, is a useful marker to indicate aberrant lipidated apolipoprotein B accumulation in the hepatocyte ER. Blockade of the ER-to-Golgi transport by either vesicle transport inhibitors or dominant-negative Arf1 caused a significant increase in ApoB-crescents. However, a low concentration of Brefeldin A induced the same result without affecting protein secretion, suggesting ADP-ribosylation as an additional mechanism. ADP-ribosylation inhibitors not only suppressed the increase of ApoB-crescents, but also rapidly dissolved existing ApoB-crescents. These results implicate the involvement of ADP-ribosylation in the ApoB-crescent formation and maintenance process at the ER.     

The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins

Guanine nucleotide exchange factors (GEFs) regulate the activity of small G proteins by catalysing the intrinsically slow exchange of GDP for GTP. The mechanism involves the formation of trimeric G protein-nucleotide-GEF complexes, followed by the release of nucleotide to form stable binary G protein-GEF complexes. A number of structural studies of G protein-GEF complexes have shown large structural changes induced in the nucleotide binding site. Together with a recent structure of a trimeric complex, these studies have suggested not only some common principles but also large differences in detail in the GEF-mediated exchange reaction. Several structures suggested that a glutamic acid residue in switch II, which is part of the DxxGQE motif and highly conserved in Ras-like G proteins, might have a decisive mechanistic role in GEF-mediated nucleotide exchange reactions. Here we show that mutation of the switch II glutamate to Ala severely impairs GEF-catalysed nucleotide exchange in most, but not all, Ras family G proteins, explaining its high sequence conservation. The residue determines the initial approach of GEF to the nucleotide-loaded G protein and does not appreciably affect the formation of a binary nucleotide-free complex. Its major effect thus appears to be the removal of the P-loop lysine from its interaction with the nucleotide.     

Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO

We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant K_D = 3.44 × 10⁻⁷ M, similar to the K_D = 3.13 × 10⁻⁷ M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser₃₉₂. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser₃₉₂ phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.