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The lysosome is a cellular signalling hub at the point of convergence of endocytic and autophagic pathways, where the contents are degraded and recycled. Pleckstrin homology domain-containing family member 1 (PLEKHM1) acts as an adaptor to facilitate the fusion of endocytic and autophagic vesicles with the lysosome. However, it is unclear how PLEKHM1 function at the lysosome is controlled. Herein, we show that PLEKHM1 co-precipitates with, and is directly phosphorylated by, mTOR. Using a phospho-specific antibody against Ser432/S435 of PLEKHM1, we show that the same motif is a direct target for ERK2-mediated phosphorylation in a growth factor-dependent manner. This dual regulation of PLEKHM1 at a highly conserved region points to a convergence of both growth factor- and amino acid-sensing pathways, placing PLEKHM1 at a critical juncture of cellular metabolism.

Baicalin is one of the main flavonoids of the dried root of Scutellaria baicalensis Georgi and is reported to exert beneficial effects on the regulation of glucose/lipid metabolism. However, understanding its specific target and unique mechanism for improving glucose utilization is a challenge. In this paper, target fishing with a baicalin probe reveals that baicalin interacts with AKT. An immunofluorescence assay further demonstrates the colocalization of baicalin with AKT in the cytoplasm. A competitive test and virtual docking show that baicalin might bind to the pleckstrin homology domain of AKT. This specific binding hampers AKT membrane translocation, activates the phosphorylation of AKT on Ser473, induces the downstream glycogen synthase kinase 3β activation, and affects glycogen synthesis.

The small GTPase Arf6 is transiently associated with the ingressing cleavage furrow and subsequently targeted to the Flemming body during cytokinesis, suggesting its activation around the cleavage furrow. Here, we show that EFA6 (exchange factor for Arf6) localizes on the cleavage furrow through its PH domain. Time-lapse analysis showed that both EFA6 and Arf6 are transiently localized around the ingressing cleavage furrow, but only Arf6 is subsequently targeted to the Flemming body. Expression of an EFA6 mutant suppresses Arf6 recruitment onto the Flemming body. These results suggest that EFA6 participates in activation of Arf6 around the cleavage furrow during cytokinesis.

The pleckstrin homology (PH) domain-containing protein casein kinase 2 interacting protein-1 (CKIP-1) plays an important role in regulation of bone formation and muscle differentiation. How CKIP-1 localization is determined remains largely unclear. We observed that isolated CKIP-1-PH domain was predominantly localized in the nucleus and the C-terminus of CKIP-1 counteracted its nuclear localization. The net charge of basic residues and a serine-rich motif within the PH domain plays a pivotal role in the localization switch of both full-length CKIP-1 and the isolated PH domain. We propose that the N-terminal PH domain and C-terminal auto-inhibitory region of CKIP-1 coordinate to determine its subcellular localization and the nucleus–plasma membrane shuttling.

Phosphatidylinositol 3-kinase pathways play key regulatory roles in cell cycle progression into S phase. In this study, we demonstrated that Akt1/PKBalpha isoform plays an essential role in G(1)/S transition and proliferation. Cells lacking Akt1/PKBalpha showed an attenuated proliferation as well as G(1)/S transition, whereas cells lacking Akt2/PKBbeta showed normal proliferation and G(1)/S transition. The effect of Akt1/PKBalpha on cell proliferation and G(1)/S transition was completely abolished by swapping pleckstrin homology (PH) domain with that of Akt2/PKBbeta. Finally, full activation of Akt/PKB and cyclin D expression was achieved by the Akt1/PKBalpha or chimeric proteins containing the PH domain of Akt1/PKBalpha indicating that the PH domain of Akt1/PKBalpha provides full kinase activity and is necessary for the G(1)/S transition.

Connector enhancer of KSR (CNK) proteins have been proposed to act as scaffolds in the Ras-MAPK pathway. In this work, using in vivo bioluminescence resonance energy transfer (BRET) assays and in vitro co-immunoprecipitation, we show that hCNK1 interacts with the active form of Rho A (G14V) proteins. The domain of hCNK1 that allows binding to Rho proteins involves the C-terminal PH domain. Overexpression of hCNK1 does not affect the actin cytoskeleton and does not modify the appearance of stress fibers in cells overexpressing a constitutively active form of RhoA. In contrast, hCNK1 was able to significantly decrease the RhoA-induced transcriptional activity of the serum response element (SRE) without effect on the Ras-induced SRE activation. These results identify hCNK1 as a specific partner of Rho proteins both in vitro and in vivo and suggest a role of hCNK1 in the signal transduction of Rho proteins.

We have previously described a partial cDNA sequence encoding a RhoGAP protein, GAP25 that is homologous to the recently reported ArhGAP9 and ArhGAP12. We now describe a related new member ArhGAP15 that shares a number of domain similarities, including a pleckstrin homology (PH) domain, a RhoGAP domain and a novel motif N-terminal to the GAP domain. This novel motif was found to be responsible for nucleotide-independent Rac1 binding. Using swop mutants of Rac/Cdc42, we have established that the binding is through the C-terminal half of Rac1. The GAP domain of ArhGAP15 showed specificity towards Rac1 in vitro. The PH domain is required for ArhGAP15 to localize to cell periphery and over-expression of the full-length ArhGAP15, but not the mutant with a partial deletion of the PH domain, resulted in an increase in actin stress fibers and cell contraction. These morphological effects can be attenuated by the co-expression of dominant negative Rac1N17. HeLa cells expressing ArhGAP15 were also resistant to phorbol myristatate acetate treatment, suggesting that ArhGAP15 is a potential regulator of Rac1.

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCμ, PKD2 and PKD3/PKCν. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.

The oxysterol binding proteins (OSBPs) are believed to control cholesterol homeostasis but their precise mechanism of action is not well understood. The Dictyostelium osbA gene encodes a predicted OSBP, OSBPa, which lacks the PH domain that in most other OSBPs directs targetting to the Golgi. OSBPa instead localises selectively to the cell periphery and also, in some cells, to the perinuclear region. OSBPa null strains form normal fruiting bodies but are defective in the regulation of the transition from slug migration to culmination. Thus a plasma membrane-enriched OSBP family member is essential for correct regulation of the slug–fruiting body switch.

The Dbl homology (DH) domain was first identified in the Dbl oncogene product as the limit region required for mediating guanine nucleotide exchange on the Rho family GTPase Cdc42. Since the initial biochemical characterization of the DH domain, this conserved motif has been identified in a large family of proteins. In each case, a pleckstrin homology (PH) domain immediately follows the DH domain and this tandem DH–PH module is the signature motif of the Dbl family of guanine nucleotide exchange factors (GEFs). Recent structural studies have provided significant insight into the molecular basis of guanine nucleotide exchange by Dbl family GEFs, opening the door for understanding the specificity of the DH/GTPase interaction as well as providing a starting point for understanding how the exchange activity of these proteins is modulated to achieve specific biological outcomes in the cell.

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.

Several guanine nucleotide exchange factors (GEFs) for Rho-GTPases have been identified, all of them containing a Dbl homology (DH) and pleckstrin homology (PH) domain, but exhibiting different specificities to the Rho family members, Rho, Rac and Cdc42. We report here that KIAA0380, a protein with a tandem DH/PH domain, an amino-terminal PDZ domain and a regulator of G protein signalling (RGS) homology domain, is a specific GEF for RhoA, but not for Rac1 and Cdc42, as determined by GDP release, guanosine 5′-O-(3-thio)triphosphate (GTPγS) binding and protein binding assays. When expressed in J82 cells, DH/PH domain-containing forms of KIAA0380 induced actin stress fibers, whereas expression of the RGS homology domain prevented lysophosphatidic acid (LPA)-induced stress fiber formation.

To elucidate the mechanism of the circadian rhythm, genes differentially expressed during subjective day and night in the rat suprachiasmatic nucleus (SCN), a circadian oscillator in mammals, were surveyed by a differential display method. We isolated a novel gene, scop (SCN circadian oscillatory protein), that was expressed in a circadian manner in the SCN. SCOP protein is predominantly expressed in the brain and has domains including a pleckstrin homology domain, leucine-rich repeats, a protein phosphatase 2C-like domain and a glutamine-rich region. The structural feature of SCOP protein suggests its role in the intracellular signaling in the SCN.

Ras-GRF is a guanine nucleotide exchange factor that activates Ras proteins. Its activity on Ras in cells is enhanced upon calcium influx. Activation follows calcium-induced binding of calmodulin to an IQ motif near the N-terminus of Ras-GRF. Ras-GRF also contains a Dbl homology (DH) domain C-terminal to the IQ motif. In many proteins, DH domains act as exchange factors for Rho-GTPase family members. However, we failed to detect exchange activity of this domain on well characterized Rho family members. Instead, we found that mutations analogous to those that block exchange activity of Dbl prevented Ras-GRF activation by calcium/calmodulin in vivo. All DH domains are followed immediately by a pleckstrin homology (PH) domain. We found that a mutation at a conserved site within the PH domain following the DH domain also prevented Ras-GRF activation by calcium in vivo. These results suggest that in addition to playing a role as activators of Rho proteins, DH domains can also contribute to the coupling of cellular signals to Ras activation.

AlF4− activates heterotrimeric G-proteins Gα subunits but not small GDP/GTP-binding proteins like ARF1. On retinal membranes containing holotransducin (GtαGDP-Gtβγ and incubated with ARFGDP, AlF4− induced GtαGDP-AlF4 release and ARFGDP binding, probably to the remaining membrane-attached Gtβγ. On phospholipid vesicles reconstitued with Gtβγ, ARFGDP bound in proportion to Gtβγ, and was released upon subsequent GtαGDP addition. Thus ARFGDP competes with GtαGDP for binding to Gtβγ, probably through a conserved motif in the ‘α2 helix’ of Gtα and ARF. This motif is found in the C-terminal helix of PH domains that bind to Gβγ.

The initial reports on pleckstrin homology (PH) domains almost 20years ago described them as sequence feature of proteins involved in signal transduction processes. Investigated at first along the phospholipid binding properties of a small subset of PH representatives, the PH fold turned out to appear as mediator of phosphotyrosine and polyproline peptide binding to other signaling proteins. While phospholipid binding now seems rather the exception among PH-like domains, protein–protein interactions established as more and more important feature of these modules. In this review we focus on the PH superfold as a versatile protein–protein interaction platform and its three-dimensional integration in an increasing number of available multidomain structures.

We describe in this report the presence of a nuclear localization signal (NLS) overlapping the calmodulin-binding domain (CaM-BD) of the growth factor receptor bound protein 7 (Grb7). We show that deletion of the CaM-BD of Grb7 prevents its nuclear localization, and that its Src homology 2 (SH2) domain might participate as well in the translocation process. Also, treating cells with the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) enhances the presence of Grb7 in the nucleus. We propose that CaM inhibits the translocation of Grb7 to the nucleus after binding to its CaM-BD and therefore occluding its overlapping NLS., This work was financed by grants (to A.V.) from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494), the European Commission (contract PITN-GA-2011-289033), and the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349). IG-P was supported by a predoctoral fellowship from the Ministerio de Ciencia e Innovación.

4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) stimulates human platelets via alpha 2A-adrenergic receptor-mediated activation of protein kinase C (PKC) independent of the phospholipase C pathway. Here we show, that in permeabilized platelets activation of PKC by DIDS (20 microM), measured as 32P incorporation in pleckstrin, is completely inhibited by guanosine 5'-(2-O-thio)diphosphate (200 microM), an inhibitor of heterotrimeric G-proteins. Also pertussin toxin (4 micrograms/ml), which ADP-ribosylates the alpha-subunits of Gi's and Go, prevents pleckstrin phosphorylation by DIDS. N-Ethylmaleimide (50 microM), which uncouples Gi from alpha 2A-adrenoceptors, inhibits pleckstrin phosphorylation by DIDS in intact platelets. Activation of PKC by 55 nM phorbol 12-myristate 13-acetate and 500 nM platelet-activating factor are not disturbed by NEM. DIDS inhibits by 40 +/- 5% (n = 4) the pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41 kDa protein fraction previously shown to contain the alpha-subunits of Gi alpha-1, Gi alpha-2 and Gi alpha-3. Thus, the alpha 2A-adrenergic receptor activates PKC via a G-protein of the Gi-family.

Phospholipase D1 and D2 (PLD1, PLD2) both have PX and PH domains in their N-terminal regions with these inositol lipid binding domains playing key roles in regulating PLD activity and localisation. The activity of PLD1 is also regulated by protein kinase C and members of the Rho and Arf families of GTPases. Each of these proteins binds to unique sites; however, there appears to be little in vitro discrimination between individual family members. In agonist-stimulated cells, however, there is specificity, with, for example in RBL-2H3 cells, antigen stimulating the activation of PLD1 by association with Arf6, Rac1 and protein kinase Cα. PLD2 appears to be less directly regulated by GTPases and rather is primarily controlled through interaction with phosphatidylinositol 4-phosphate 5-kinase that generates the activating phosphatidylinositol 4,5-bisphosphate.

PHIP was isolated as an insulin receptor substrate 1 (IRS-1) interacting protein. To date, the physiological roles of PHIP remain unknown. Here we show that mice lacking PHIP1, the full-length isoform of PHIP, are born at normal size but suffer a 40% growth deficit by weaning. PHIP1 mutant mice develop hypoglycemia and have an average lifespan of 4–5weeks. PHIP1-deficient mouse embryonic fibroblasts (MEFs) grow markedly slower than wild-type MEFs, but exhibit normal AKT phosphorylation and an increased cell proliferation in response to IGF-1 treatment. Together these results suggest that PHIP1 regulates postnatal growth in an IGF-1/AKT pathway-independent manner.

International audience; Phosphatidylinositol 5-phosphate (PtdIns5P) is emerging as a potential lipid messenger involved in several cell types, from plants to mammals. Expression of IpgD, a PtdIns(4,5)P(2) 4-phosphatase induces Src kinase and Akt, but not ERK activation and enhances interleukin II promoter activity in T-cells. Expression of a new PtdIns5P interacting domain blocks IpgD-induced T-cell activation and selective signaling molecules downstream of TCR triggering. Altogether, these data suggest that PtdIns5P may play a sensor function in setting the threshold of T-cell activation and contributing to maintain T-cell homeostasis.

Immune cells navigate through different environments where they experience different mechanical forces. Responses to external forces are determined by the mechanical properties of a cell and they depend to a large extent on the actin-rich cell cortex. We report here that Myo1G, a previously uncharacterised member of class I myosins, is expressed specifically in haematopoietic tissues and cells. It is associated with the plasma membrane. This association is dependent on a conserved PH-domain-like myosin I tail homology motif and the head domain. However, the head domain does not need to be a functional motor. Knockdown of Myo1G in Jurkat cells decreased cell elasticity significantly. We propose that Myo1G regulates cell elasticity by deformations of the actin network at the cell cortex.Structured summaryMINT-7307273: MYO1G (uniprotkb:B0I1T2) and Actin (uniprotkb:P60709) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7307283: TfR (uniprotkb:P02786) and MYO1G (uniprotkb:B0I1T2) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)

Insulin stimulation of Glut 4 translocation requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase) but the downstream pathway remains ill-defined. We demonstrated that the overexpression of PDK1 (3-phosphoinositide-dependent protein kinase 1), a downstream effector of PI 3-kinase, stimulated Glut 4 translocation in adipocytes. This effect does not require the PH domain of PDK1, but expression of the pleckstrin homology domain-deleted PDK1 inhibits the effect of insulin, but not okadaic acid, on Glut 4 translocation. These results support a role of the PDK1 pathway in the transmission of insulin signal to Glut translocation.

Plant G proteins of the ROP/RAC family regulate cellular processes including cytoskeletal rearrangement in polar growth. Activation of the ROP molecular switch is triggered by guanine nucleotide exchange factors. Plant-specific RopGEFs are exclusively active on ROPs despite their high homology to animal Rho proteins. Based on a sequence comparison of ROPs vs. animal Rho proteins together with structural data on distinct ROPs, we identified unique substrate determinants of RopGEF specificity by mutational analysis: asparagine 68 next to switch II, arginine 76 of a putative phosphorylation motif and the Rho insert are essential for substrate recognition by RopGEFs. These data also provide first evidence for a function of the Rho insert in interactions with GEFs.

Membrane rafts are regions of increased lipid acyl chain order that differ in their lipid and protein composition from the surrounding membrane. By providing an additional level of compartmentalization they have been proposed to serve many functions in cellular signal transduction and trafficking. We will review their potential involvement in different forms of membrane traffic, explicitly excluding signalling, and discuss select aspects of the raft hypothesis in its current form.

Inducible T-cell kinase (ITK) is a member of the Tec family of tyrosine kinases that are involved in signals emanating from cytokine receptors, antigen receptors and other lymphoid cell surface receptors. Stimulation of tyrosine phosphorylation and activation of ITK by the T-cell antigen receptor, CD28 and CD2 requires the presence of the Src family kinase Lck in T-cells. We have previously demonstrated that the activation of ITK by Src family kinases uses a phosphatidylinositol 3-kinase pathway, which recruits ITK to the membrane via its pleckstrin homology (PH) domain where it is acted upon by Src. We have further explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that deletion of the proline rich sequence found in the Tec homology domain of ITK results in reduced basal activity of ITK approximately 50%. These differences in the basal activity of ITK were observed when the PH domain was deleted or when the kinase was membrane targeted. Furthermore, this deletion reduces the ability of the Src family kinase Lck to activate ITK, as well as to induce the ITK mediated tyrosine phosphorylation of its substrate PLCγ1. By contrast, deletion of the SH3 domain of ITK resulted in a two-fold increase in the basal activity of ITK, and allowed this mutant to have an enhanced response to Lck. These results suggest that the proline rich region within the Tec homology domain of ITK regulates its basal activity and its response to Src family kinase signals.

A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the Gα12 family stimulate Rho-dependent signaling pathways.

The pleckstrin homology (PH) domain of the protooncogenic serine/threonine protein kinase PKB/Akt can bind phosphoinositides. A yeast-based two-hybrid system was employed which identified inosine-5′ monophosphate dehydrogenase (IMPDH) type II as specifically interacting with PKB/Akts PH domain. IMPDH catalyzes the rate-limiting step of de novo guanosine-triphosphate (GTP) biosynthesis. Using purified fusion proteins, PKB/Akts PH domain and IMPDH associated in vitro and this association moderately activated IMPDH. Purified PKB/Akt also associated with IMPDH in vitro. We could specifically pull-down PKB/Akt or IMPDH from mammalian cell lysates using glutathione-S-transferase (GST)–IMPDH or GST–PH domain fusion proteins, respectively. Additionally, PKB/Akt and IMPDH could be co-immunoprecipitated from COS cell lysates and active PKB/Akt could phosphorylate IMPDH in vitro. These results implicate PKB/Akt in the regulation of GTP biosynthesis through its interaction with IMPDH, which is involved in providing the GTP pool used by signal transducing G-proteins.

Aarskog–Scott Syndrome (AAS) is an X-linked disorder characterised by short stature and multiple facial, limb and genital abnormalities. A gene, FGD1, altered in a patient with AAS phenotype, has been identified and found to encode a protein with homology to Rho/Rac guanine nucleotide exchange factors (Rho/Rac GEF). However, since this original report on identification of a mutated FGD1 gene in an AAS patient, no additional mutations in the FGD1 gene have been described. We analysed 13 independent patients with clinical diagnosis of AAS. One patient presented a mutation that results in a nucleotide change in exon 10 of the FGD1 gene (G2559>A) substituting a Gln for Arg in position 610. The mutation was found to segregate with the AAS phenotype in affected males and carrier females in the family of this patient. Interestingly, Arg-610 is located within one of the two pleckstrin homology (PH) domains of the FGD1 gene and it corresponds to a highly conserved residue which has been involved in InsP binding in PH domains of other proteins. The same residue is often mutated in the Bruton’s tyrosine kinase (Btk) gene in patients with an X-linked agammaglobulinemia. The Arg610Gln mutation represents the first case of a mutation in the PH domain of the FGD1 gene and additional evidence that mutations in PH domains can be associated to human diseases.

Mammalian Vav signal transducer protein couples tyrosine kinase signals with the activation of the Rho/Rac GTPases, thus leading to cell differentiation and/or proliferation. We have isolated and characterized the DroVav gene, the homologue of hVav in Drosophila melanogaster. DroVav encodes a protein (793 residues) whose similarity with hVav is 47% and with hVav2 and hVav3 is 45%. DroVav preserves the unique, complex structure of hVav proteins, including the ‘calponin homology’, dbl homology, pleckstrin homology; SH2 and SH3 domains in addition to regions that are acidic rich, proline rich and cysteine rich. DroVav is located on the X chromosome in polytene interval 18A5;18B and is expressed in all stages of development and in all tissues. In mammalian cells, DroVav is tyrosine-phosphorylated in response to epidermal growth factor receptor (EGFR) induction; in vitro, the DroVav SH2 region is associated with tyrosine-phosphorylated EGFR. Thus, DroVav probably plays a pivotal role as a signal transducer protein during fruit fly development.

The Ras-GRF1 exchange factor molecule contains in addition to the catalytic domain two pleckstrin homology (PH1 and PH2), one IQ and one Dbl homology (DH) domains. In this study we investigated the role of such additional domains. We found that a Ras-GRF1 mutant lacking PH1 and IQ domains is sufficient to activate c-fos promoter in response to lysophosphatidic acid (LPA). The same mutant did not increase external stimuli-regulated kinase (ERK) activity, suggesting an additional mechanism for the induction of gene transcription. Isolated DH-PH2 module activates c-Jun NH2-terminal kinase and the c-fos promoter in response to LPA, providing the basis for an ERK-independent mechanism. These results provide evidence that Ras-GRF1 acts as a bifunctional molecule on both ERK-dependent and independent pathways.

Distinct forms of inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates. SHIP1 is the 145-kDa SH2 domain-containing inositol 5-phosphatase expressed in haematopoietic cells. SHIP2 is a related but distinct gene product. We report here that SHIP2 can be expressed in an active form both in Escherichia coli and in COS-7 cells. A truncated 103-kDa recombinant protein could be purified from bacteria that display both inositol 1,3,4,5-tetrakisphosphate (InsP4) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) phosphatase activities. COS-7 cell lysates transfected with SHIP2 had increased PtdIns(3,4,5)P3 phosphatase activity as compared to the vector alone.

Pleckstrin homology (PH) domains are discrete structural modules present in numerous proteins involved in signal transduction processes. In the case of the beta-adrenergic receptor kinase (betaARK), PH domain-mediated binding of two ligands, the betagamma subunits of heterotrimeric G proteins (Gbetagamma) and phosphatidylinositol 4,5-bisphosphate (PIP2), has been shown to be required for the kinase function. In this study, the ability of Gbetagamma and PIP2 to affect membrane localization of betaARK is used to define the ligand binding characteristics of the betaARK PH domain. The binding of these ligands to the PH domain of the intact kinase is shown to be cooperative, Gbetagamma increasing the affinity of the PH domain for PIP2. Notably, although PIP2-dependent membrane association of betaARK is observed at high concentrations of this lipid, in the absence of Gbetagamma, no receptor phosphorylation is observed. Peptides derived from the receptor intracellular loop inhibit the receptor phosphorylation without affecting the membrane translocation of the kinase complex, suggesting that betaARK activity does not necessarily correlate with the amount of betaARK associated with the membrane. These results point to a distinct role for each PH domain ligand in betaARK-mediated receptor phosphorylation. Strikingly, the ligand binding characteristics of the isolated betaARK PH domain fused to glutathione S-transferase are very different from those of the PH domain of the intact kinase. Thus, in contrast to the native protein, the isolated PH domain binds Gbetagamma and PIP2 independently and with no apparent cooperativity. That protein environment plays an important role in determining the ligand binding characteristics of a particular PH domain highlights the potential risks of inferring mechanisms from studies of isolated PH domains.

We have identified, characterized and cloned human, mouse and chicken cDNA of a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. We subsequently named it YAP for Yes-associated protein. Analysis of the YAP sequence revealed a protein module that was found in various structural, regulatory and signaling molecules. Because one of the prominent features of this sequence motif is the presence of two conserved tryptophans (W), we named it the WW domain. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous proline-rich region. Binding assays and site-specific mutagenesis have shown that the proline-rich motif binds with relatively high affinity and specificity to the WW domain of YAP, with a preliminary consensus that is different from the SH3-binding PXXP motif. This suggests that the WW domain has a role in mediating protein-protein interactions via proline-rich regions, similar but distinct from Src homology 3 (SH3) domains. Based on this finding, we hypothesize that additional protein modules exist and that they could be isolated using proline-rich peptides as functional probes.

The pleckstrin homology (PH) domain is extended in the Btk kinase family by a region designated the TH (Tec homology) domain, which consists of about 80 residues preceding the SH3 domain. The TH domain contains a conserved 27 amino acid stretch designated the Btk motif and a proline-rich region. Sequence similarity was found to a putative Ras GTPase activating protein and a human interferon-γ binding protein both in the PH domain and the Btk motif region. SLK1/SSP31 protein kinase and a non-catalytic p85 subunit of PI-3 kinase had similarity only with the proline rich region. The identification of a PH domain extension in some signal transduction proteins in different species suggests that this region is involved in protein—protein interactions.

Phospholipase D was first described in plant tissue but has recently been shown to occur in mammalian cells where it is activated by cell surface receptors. Its mode of activation by receptors in unclear. Biochemical studies suggest that it may occur downstream of other effector proteins and that small GTP-dependent regulatory proteins may be involved. The sequence in a non-designated region of mammalian phospholipase D1 and 2 shows similarity to a structural domain that is present in signalling proteins that are regulated by protein kinases or heterotrimeric G-proteins. Mammalian phospholipase D has structural similarities with other lipid signalling phospholipases and thus may be regulated by receptors in an analogous fashion.

The recently described Spred protein family has been implicated in the modulation of receptor tyrosine kinase signalling. We report the crystal structure of the Enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain from Xenopus tropicalis Spred1, solved to 1.15 A resolution. This structure confirms that the Spred EVH1 adopts the pleckstrin-homology fold, with a similar secondary structure to Enabled. A translation of one of the peptide-binding groove β-strands narrows this groove, whilst one end of the groove shows structural flexibility. We propose that Spred1 will bind peptides that are less proline-rich than other EVH1 domains, with conformational changes indicating an induced fit.

A plant homologue of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3-phosphoinositide-dependent protein kinase-1. Like the mammalian 3-phosphoinositide-dependent protein kinase-1, Arabidopsis 3-phosphoinositide-dependent protein kinase-1 and rice 3-phosphoinositide-dependent protein kinase-1 possess a kinase domain at N-termini and a pleckstrin homology domain at their C-termini. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3-phosphoinositide-dependent protein kinase-1 homologues. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is able to activate human protein kinase Bα (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3-phosphoinositides.

We have cloned a novel unconventional myosin gene myoM in Dictyostelium. Phylogenetic analysis of the motor domain indicated that MyoM does not belong to any known subclass of the myosin superfamily. Following the motor domain, two calmodulin-binding IQ motifs, a putative coiled-coil region, and a Pro, Ser and Thr-rich domain, lies a combination of dbl homology and pleckstrin homology domains. These are conserved in Rho GDP/GTP exchange factors (RhoGEFs). We have identified for the first time the RhoGEF domain in the myosin sequences. The growth and terminal developmental phenotype of Dictyostelium cells were not affected by the myoM− mutation. Green fluorescent protein-tagged MyoM, however, accumulated at crown-shaped projections and membranes of phase lucent vesicles in growing cells, suggesting its possible roles in macropinocytosis.

Various drugs such as phenobarbital (PB) trigger translocation of constitutive active/adrostane receptor (CAR) from the cytoplasm into the nucleus of mouse liver cells without directly binding to the receptor. We have now characterized the guanine nucleotide exchange factor epithelial cell-transforming gene 2 (ECT2) as a PB-inducible factor as well as a cellular signal that represses PB-triggered nuclear translocation of CAR. When CFP-tagged ECT2 was co-expressed with YFP-tagged CAR in the liver of Car−/− mice, ECT2 repressed CAR nuclear translocation. Coexpression of various deletion mutants delineated this repressive activity to the tandem Dbl homology/pleckstrin homology domains of ECT2 and to their cytosolic expression. CAR directly bound to the PH domain. Thus, ECT2 may comprise a part of the PB response signal regulating the intracellular trafficking of CAR.

Pleckstrin homology (PH) domains are protein modules found in proteins involved in many cellular processes. The majority of PH domain-containing proteins require membrane association for their function. It has been shown that most PH domains interact directly with the cell membrane by binding to phosphoinositides with a broad range of specificity and affinity. While a highly specific binding of the PH domain to a phosphoinositide can be necessary and sufficient for the correct recruitment of the host protein to the membrane, a weaker and less specific interaction may be necessary but not sufficient, thus probably requiring alternative, co-operative mechanisms.

The pleckstrin homology and C2 domains are modular protein structures involved in mediating intermolecular interactions. Although they represent distinct domains, there are several parallels regarding their function and type of interactions in which they participate. Both domains are stable structural entities that incorporate variable regions which, in different proteins, can be adapted to perform a specific function through binding to membrane phospholipids or specific protein ligands. A number of recent examples illustrate the function of some of these domains in regulated membrane attachment, with an important role in many cellular signalling pathways.

Affixin/β-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK αp21-activated kinase (PAK)-interactive exchange factor (PIX) and βPIX accumulate. The association of affixin and βPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant βPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through α and βPIXs in skeletal muscle. Structured summary MINT-6179203, MINT-6179212, MINT-6178859, MINT-6178812, MINT-6178832, MINT-6178843: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by coimmunoprecipitation (MI:0019) MINT-6179221: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with αpix (uniprotkb:Q8K4I3) by coimmunoprecipitation (MI:0019) MINT-6178962, MINT-6178983: Affixin (uniprotkb:Q9HBI1) physically interacts (MI:0218) with βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) MINT-6179002, MINT-6179021: Affixin (uniprotkb:Q9HBI1) binds (MI:0407) βpix (uniprotkb:Q9ES28) by pull-down (MI:0096) MINT-6179039: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Rac1 (uniprotkb:P63001) by pull-down (MI:0096) MINT-6179054: PAK1 (uniprotkb:Q13153) physically interacts (MI:0218) with Cdc42 (uniprotkb:P70766) by pull-down (MI:0096) MINT-6178790: Affixin (uniprotkb:Q9HBI1) and αpix (uniprotkb:Q8K4I3) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-6178760: Affixin (uniprotkb:Q9HBI1) and βpix (uniprotkb:Q9ES28) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-6178801: Affixin (uniprotkb:Q9HBI1) and dysferlin (uniprotkb:Q9ESD7) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-6178779: Affixin (uniprotkb:Q9HBI1) and ILK (uniprotkb:O55222) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Pleckstrin homology (PH) domains are 100–120 amino acid protein modules best known for their ability to bind phosphoinositides. All possess an identical core β-sandwich fold and display marked electrostatic sidedness. The binding site for phosphoinositides lies in the center of the positively charged face. In some cases this binding site is well defined, allowing highly specific and strong ligand binding. In several of these cases the PH domains specifically recognize 3-phosphorylated phosphoinositides, allowing them to drive membrane recruitment in response to phosphatidylinositol 3-kinase activation. Examples of these PH domain-containing proteins include certain Dbl family guanine nucleotide exchange factors, protein kinase B, PhdA, and pleckstrin-2. PH domain-mediated membrane recruitment of these proteins contributes to regulated actin assembly and cell polarization. Many other PH domain-containing cytoskeletal proteins, such as spectrin, have PH domains that bind weakly, and to all phosphoinositides. In these cases, the individual phosphoinositide interactions may not be sufficient for membrane association, but appear to require self-assembly of their host protein and/or cooperation with other anchoring motifs within the same molecule to drive membrane attachment.

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing RhoGAP1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.

Members of the Cool protein family contain SH3, Dbl, and pleckstrin homology domains and are binding partners for the p21-activated kinase (PAK). Using the yeast two-hybrid screen, we identified Cbl-b as a Cool family binding partner. We co-immunoprecipitated endogenous Cool and Cbl-b from a variety of breast cancer cell lines. The Cool–Cbl-b interaction requires the SH3 domain of Cool and competes with the binding of PAK to Cool proteins. Expression of Cbl-b effectively blocks the ability of Cool-2 to stimulate PAK, thus providing an additional mechanism, aside from catalyzing receptor ubiquitination, by which Cbl-b acts as a negative regulator for signaling activities requiring PAK activation.

The inositol lipid kinases were investigated in the cytoskeletons of human platelets. In the absence of added lipids the kinases were only barely detectable in the Triton-soluble fractions and undetectable in cytoskeletons of resting cells. However at least 30% of the total phosphatidylinositol kinase was present in the cytoskeleton as revealed by saturation of the enzyme. Phosphatidylinositol 4-phosphate kinase was also found in significant amounts in the cytoskeletons. On the other hand, both enzymes being only recovered in the particulate fraction of the cells, we suggest that inositol lipid kinases may be present near the anchoring points of the cytoskeletons at the membranes.