Identification of myosin II as a binding protein to the PH domain of protein kinase B

Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a Kd value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB.     

AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.     

Tec homology (TH) adjacent to the PH domain

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.     

The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors

Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin-like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin- and/or IGF1-mediated signaling.     

Structure basis and unconventional lipid membrane binding properties of the PH-C1 tandem of rho kinases

Rho kinase (ROCK), a downstream effector of Rho GTPase, is a serine/threonine protein kinase that regulates many crucial cellular processes via control of cytoskeletal structures. The C-terminal PH-C1 tandem of ROCKs has been implicated to play an autoinhibitory role by sequestering the N-terminal kinase domain and reducing its kinase activity. The binding of lipids to the pleckstrin homology (PH) domain not only regulates the localization of the protein but also releases the kinase domain from the close conformation and thereby activates its kinase activity. However, the molecular mechanism governing the ROCK PH-C1 tandem-mediated lipid membrane interaction is not known. In this study, we demonstrate that ROCK is a new member of the split PH domain family of proteins. The ROCK split PH domain folds into a canonical PH domain structure. The insertion of the atypical C1 domain in the middle does not alter the structure of the PH domain. We further show that the C1 domain of ROCK lacks the diacylglycerol/phorbol ester binding pocket seen in other canonical C1 domains. Instead, the inserted C1 domain and the PH domain function cooperatively in binding to membrane bilayers via the unconventional positively charged surfaces on each domain. Finally, the analysis of all split PH domains with known structures indicates that split PH domains represent a unique class of tandem protein modules, each possessing distinct structural and functional features.     

Ceramide kinase targeting and activity determined by its N-terminal pleckstrin homology domain

The N-terminus of ceramide kinase (CERK) is thought to be myristoylated and to contain a pleckstrin homology (PH) domain. We found that deletion of this region (DeltaPH-CERK) ablates activity. This is not due to prevention of N-terminal myristoylation since a G2A CERK mutant, which cannot be myristoylated, was active. CERK was able to bind liposomes, as well as the isolated unmyristoylated PH domain; DeltaPH-CERK was not. Upon analysis of EGFP-tagged proteins, CERK was found associated with the Golgi complex. Osmotic cell swelling induced translocation of CERK to the plasma membrane and formation of large vesicles displaying bound CERK. None of these features occurred with DeltaPH-CERK, which remained disseminated throughout the cytoplasm. These findings show that the PH domain of CERK is essential for localization, translocation, and activity of this lipid kinase.     

Crystal Structure of the Pleckstrin Homology Domain from the Ceramide Transfer Protein: Implications for Conformational Change upon Ligand Binding

Ceramide transfer protein (CERT) is responsible for the nonvesicular trafficking of ceramide from the endoplasmic reticulum (ER) to the trans Golgi network where it is converted to sphingomyelin (SM). The N-terminal pleckstrin homology (PH) domain is required for Golgi targeting of CERT by recognizing the phosphatidylinositol 4-phosphate (PtdIns(4)P) enriched in the Golgi membrane. We report a crystal structure of the CERT PH domain. This structure contains a sulfate that is hydrogen bonded with residues in the canonical ligand-binding pocket of PH domains. Our nuclear magnetic resonance (NMR) chemical shift perturbation (CSP) analyses show sulfate association with CERT PH protein resembles that of PtdIns(4)P, suggesting that the sulfate bound structure likely mimics the holo form of CERT PH protein. Comparison of the sulfate bound structure with the apo form solution structure shows structural rearrangements likely occur upon ligand binding, suggesting conformational flexibility in the ligand-binding pocket. This structural flexibility likely explains CERT PH domain’s low affinity for PtdIns(4)P, a property that is distinct from many other PH domains that bind to their phosphoinositide ligands tightly. This unique structural feature of CERT PH domain is probably tailored towards the transfer activity of CERT protein where it needs to shuttle between ER and Golgi and therefore requires short resident time on ER and Golgi membranes.     

Human Sin1 contains Ras-binding and pleckstrin homology domains and suppresses Ras signalling

Human Sin1 (SAPK-interacting protein 1) is a member of a conserved family of orthologous proteins that have an essential role in signal transduction in yeast and Dictyostelium. This study demonstrates that most Sin1 orthologues contain both a Raf-like Ras-binding domain (RBD) and a pleckstrin homology (PH) domain. These domains are functional in the human Sin1 protein, with the PH domain involved in lipid and membrane binding by Sin1, and the RBD able to bind activated H-and K-Ras. Sin1 and Ras co-immunoprecipitated and co-localised, showing that these proteins associate with each other in vivo. Overexpression of Sin1 inhibited the activation of ERK, Akt and JNK signalling pathways by Ras. In contrast, siRNA knockdown of endogenous Sin1 protein expression in HEK293 cells enhanced the activation of ERK1/2 by Ras. These data suggest that Sin1 is a mammalian Ras-inhibitor.     

RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro.

The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.     

PKB/Akt interacts with inosine-5' monophosphate dehydrogenase through its pleckstrin homology domain

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.     

Characterization of a novel member of the DOK family that binds and modulates Abl signaling

A novel member of the p62(dok) family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62(dok) bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62(dok), DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62(dok), suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.     

Cloning and characterization of PHIP, a novel insulin receptor substrate-1 pleckstrin homology domain interacting protein

Insulin receptor substrate-1 (IRS-1) protein is a major substrate of the insulin receptor tyrosine kinase and is essential for transducing many of the biological effects of insulin including mitogenesis, gene expression, and glucose transport. The N terminus of IRS-1 contains a pleckstrin homology (PH) domain that is critical for recognition and subsequent phosphorylation of IRS-1 by the activated insulin receptor. Here we report the isolation of a novel protein, PHIP (PH-interacting protein), which selectively binds to the PH domain of IRS-1 in vitro and stably associates with IRS-1 in vivo. Importantly, mutants of the IRS-1 PH domain that disrupt the PH fold fail to bind to PHIP. Anti-phosphotyrosine immunoblots of PHIP revealed no discernible insulin receptor-regulated phosphorylation, suggesting that PHIP is not itself a substrate of the insulin receptor. In contrast to full-length PHIP, overexpression of the PH-binding region of PHIP has a pronounced inhibitory effect on insulin-induced IRS-1 tyrosine phosphorylation levels. Furthermore, expression of this dominant-negative PHIP mutant leads to a marked attenuation of insulin-stimulated mitogen-activated protein kinase activity. We conclude that PHIP represents a novel protein ligand of the IRS-1 PH domain that may serve to link IRS-1 to the insulin receptor.     

The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ~100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.     

Cleavage and release of a soluble form of the receptor tyrosine kinase ARK in vitro and in vivo

The receptor tyrosine kinase ARK (also called AXL or UFO) is the murine prototype of a small family of receptors with an extracellular domain resembling cell adhesion molecules and a conserved tyrosine kinase domain. ARK is capable of homophilic binding, as well as of binding to GAS6, a secreted member of the class of vitamin K dependent proteins whose expression is up-regulated in growth-arrested cells. To gain understanding of the physiological role of ARK signaling, we have investigated the ARK forms which are expressed by cells in culture as well as by mouse organs. We found that ARK is not only expressed as a transmembrane protein, but is also cleaved in the extracellular domain to generate a soluble ARK form of about 65 kDa, which is easily detected in conditioned media of ARK expressing cells, in serum and plasma and in mouse organs. Soluble ARK is also produced by tumor cells in vivo. The function of these molecules could be that of binding GAS6, thereby inhibiting the interaction of this ligand with its cell-associated receptor, or they could be involved in binding to ARK itself. © 1996 Wiley-Liss, Inc.     

Pleckstrin induces cytoskeletal reorganization via a Rac-dependent pathway.

Pleckstrin homology (PH) domains are present in over one hundred signaling molecules, where they are thought to mediate membrane targeting by binding to phosphoinositides. They were initially defined at the NH(2) and COOH termini of the molecule, pleckstrin, a major substrate for protein kinase C in platelets. We have previously reported that pleckstrin associates with the plasma membrane, where it induces the formation of villous and ruffled structures from the surface of transfected cells (1). We now show that overexpression of pleckstrin results in reorganization of the actin cytoskeleton. This pleckstrin effect is regulated by its phosphorylation and requires the NH(2)-terminal, but not the COOH-terminal, PH domain. Overexpression of the NH(2)-terminal PH domain alone of pleckstrin is sufficient to induce the cytoskeletal effects. Pleckstrin-induced actin rearrangements are not inhibited by pharmacologic inhibition of phosphatidylinositol 3-kinase, nor are they blocked by co-expression of a dominant negative phosphatidylinositol 3-kinase. The cytoskeletal effects of pleckstrin can be blocked by co-expression of a dominant negative Rac1 variant, but not wild-type Rac and not a dominant negative Cdc42 variant. These data indicate that the NH(2)-terminal PH domain of pleckstrin induces reorganization of the actin cytoskeleton via a pathway dependent on Rac but independent of Cdc42 and phosphatidylinositol 3-kinase.     

Pleckstrin homology domain 1 of mouse alpha 1-syntrophin binds phosphatidylinositol 4,5-bisphosphate

Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to bind phosphatidylinositol 4, 5-bisphosphate (PtdIns4,5P2). Half-maximal binding occurred at 1.9 microM PtdIns4,5P2 and when 1.2 PtdIns4,5P2 were added per syntrophin. Binding was specific for PtdIns4,5P2 and did not occur with six other tested lipids including the similar phosphatidylinositol 4-phosphate. Binding was localized to the N-terminal pleckstrin homology domain (PH1); the second, C-terminal PH2 domain did not bind lipids. Key residues in PtdIns4,5P2 binding to a PH domain were found to be conserved in alpha-syntrophins' PH1 domains and absent in PH2 domains, suggesting a molecular basis for binding.     

The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4-phosphate

Signal transduction pathways that co-regulate a given biological process often are organized into networks by molecules that act as coincidence detectors. Phosphoinositides and the Rho-type GTPase Cdc42 regulate overlapping processes in all eukaryotic cells. However, the coincidence detectors that link these pathways into networks remain unknown. Here we show that the p21-activated protein kinase-related kinase Cla4 of yeast integrates signaling by Cdc42 and phosphatidylinositol 4-phosphate (PI4P). We found that the Cla4 pleckstrin homology (PH) domain binds in vitro to several phosphoinositide species. To determine which phosphoinositides regulate Cla4 in vivo, we analyzed phosphatidylinositol kinase mutants (stt4, mss4, and pik1). This indicated that the plasma membrane pool of PI4P, but not phosphatidylinositol 4,5-bisphosphate or the Golgi pool of PI4P, is required for localization of Cla4 to sites of polarized growth. A combination of the Cdc42-binding and PH domains of Cla4 was necessary and sufficient for localization to sites of polarized growth. Point mutations affecting either domain impaired the ability of Cla4 to regulate cell morphogenesis and the mitotic exit network (localization of Lte1). Therefore, Cla4 must retain the ability to bind both Cdc42 and phosphoinositides, the hallmark of a coincidence detector. PI4P may recruit Cla4 to the plasma membrane where Cdc42 activates its kinase activity and refines its localization to cortical sites of polarized growth. In mammalian cells, the myotonic dystrophy-related Cdc42-binding kinase possesses p21-binding and PH domains, suggesting that this kinase may be a coincidence detector of signaling by Cdc42 and phosphoinositides.     

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P₃; K_D ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P₃ as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P₂ on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P₃ is affected by the presence of PO₄ ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.     

Split pleckstrin homology domain-mediated cytoplasmic-nuclear localization of PI3-kinase enhancer GTPase

Cytoplasm-nucleus shuttling of phosphoinositol 3-kinase enhancer (PIKE) is known to correlate directly with its cellular functions. However, the molecular mechanism governing this shuttling is not known. In this work, we demonstrate that PIKE is a new member of split pleckstrin homology (PH) domain-containing proteins. The structure solved in this work reveals that the PIKE PH domain is split into halves by a positively charged nuclear localization sequence. The PIKE PH domain binds to the head groups of di- and triphosphoinositides with similar affinities. Lipid membrane binding of the PIKE PH domain is further enhanced by the positively charged nuclear localization sequence, which is juxtaposed to the phosphoinositide head group-binding pocket of the domain. We demonstrate that the cytoplasmic-nuclear shuttling of PIKE is dynamically regulated by the balancing actions of the lipid-binding property of both the split PH domain and the nuclear targeting function of its nuclear localization sequence.     

The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.