PH结构域研究进展

ＰＨ结构域是一种新发现的约由１００￣１２０个氨基酸残基组成的功能性结构域,广泛分布于单细胞生物和无脊椎、脊椎动物及人的细胞骨架蛋白和信号分子等１００多种蛋白中,能与脂类、Ｇ蛋白的βγ亚单位、ＰＫＣ等配体相结合,推测其对蛋白质具有细胞和亚细胞水平的膜定位作用,并参与调节宿主蛋白 活性,从而介导信号转导过程中的蛋白南－脂类、蛋白质－蛋白质之间的相互作用。     

Phosphoproteomic analysis identifies insulin enhancement of discoidin domain receptor 2 phosphorylation

The discoidin domain receptors (DDRs) are collagen binding receptor tyrosine kinases that play important roles in cell migration, invasion and adhesion. Crosstalk between growth factor signaling and components of the extracellular matrix are drivers of cellular function but the integrated signaling networks downstream of such crosstalk events have not been extensively characterized. In this report, we have employed mass spectrometry-based quantitative phosphotyrosine analysis to identify crosstalk between DDR2 and the insulin receptor. Our phosphoproteomic analysis reveals a cluster of phosphorylation sites in which collagen and insulin cooperate to enhance phosphotyrosine levels. Importantly, Y740 on the DDR2 catalytic loop was found in this cluster indicating that insulin acts to promote collagen I signaling by increasing the activity of DDR2. Furthermore, we identify two additional migration associated proteins that are candidate substrates downstream of DDR2 activation. Our data suggests that insulin promotes collagen I signaling through the upregulation of DDR2 phosphorylation which may have important consequences in DDR2 function in health and disease.     

Role of pleckstrin homology domain in regulating membrane targeting and metabolic function of insulin receptor substrate 3

The insulin receptor phosphorylates insulin receptor substrate (IRS) proteins on multiple tyrosine residues that act as docking sites to recruit a number of downstream signaling molecules. Here we show that IRS3 is localized both at the plasma membrane and in the nucleus. Interestingly, the nuclear localization of the protein is restricted to specific regions involved in mRNA processing and known as speckles. By using different truncated versions of the protein, we demonstrate that the pleckstrin homology (PH) domain is involved in IRS3 localization at the level of both plasma membrane and nucleus. To our knowledge this is the first report of a PH domain responsible for a nuclear targeting of the host protein. By site-directed mutagenesis, we identify residues within the PH domain critical for proper localization of IRS3. Mutations within the PH domain preventing IRS3 intracellular localization result in an inhibition of IRS3-induced glucose uptake. We conclude that the PH domain is required for IRS3 intracellular localization and, furthermore, that it has a key role in metabolic functions of IRS3. In particular, our data suggest that IRS3 intracellular localization at the plasma membrane and in the nucleus is the result of two different cooperative mechanisms both involving the PH domain.     

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.     

Structural characterization of the split pleckstrin homology domain in phospholipase C-gamma1 and its interaction with TRPC3

Phospholipase C (PLC)-gamma is unique among the PLC enzymes because each PLC-gamma isozyme contains a split pleckstrin homology (PH) domain with an SH2SH2SH3 tandem repeat insertion (where SH indicates Src homology domain) in the middle of its sequence. Split PH domains exist in a number of other proteins that play crucial signaling roles. However, little is known about the structure and function of split PH domains. The C-terminal half of the PLC-gamma split PH domain has been implicated to interact directly with the TRPC3 calcium channel, thereby providing a direct coupling mechanism between PLC-gamma and agonist-induced calcium entry. However, this interaction has not been proved by direct biochemical or structural studies. Here we determined the three-dimensional structure of the split PH domain of PLC-gamma1, and we found that the split PH domain of the enzyme folds into a canonical PH domain fold with high thermostability. The SH2SH2SH3 insertion between the beta3 and beta4 strands does not change the structure of the split PH domain. In contrast to the majority of phospholipid-binding PH domains, the PLC-gamma1 split PH domain lacks the signature lipid-binding motif located between the beta1 and beta2 strands. Consistent with this structural feature, the split PH domain of PLC-gamma1 does not bind to phospholipids. Multiple biochemical and biophysical experiments have argued against a direct interaction between TRPC3 and the C-terminal half of the PLC-gamma1 split PH domain. Our data pointed to the existence of a yet to be elucidated interaction mechanism between TRPC3 and PLC-gamma1.     

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.     

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.     

rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

Identification of p115 as a PLCgamma1-binding protein and the role of Src homology domains of PLCgamma1 in the vesicular transport

In order to gain further insight into the function(s) of PLCgamma1, we tried to identify the binding partners that can interact with the SH223 domains of PLCgamma1. Immunoscreening was performed with the purified antisera that are specific to SH223-binding proteins. Several immunoreactive clones were identified as the putative binding proteins and one of them was identified as p115. p115 was reported to be required for transcytotic fusion and subsequent binding of the vesicles to the target membrane. The interaction between PLCgamma1 and p115 was specific to carboxyl-terminal SH2 domain and SH3 domain of PLCgamma1, and also confirmed by biochemical approaches such as co-immunoprecipitation, pull-down assay, and glycerol gradient fractionation. To further characterize the role of SH domains of PLCgamma1 in the vesicle transport pathway, secreted form of alkaline phosphatase (SEAP) reporter assay was carried out. When the SH2 and/or SH3 domains of PLCgamma1 were deleted, the secretion of SEAP was significantly reduced. These findings indicate that the SH2 and SH3 domains of PLCgamma1 may play a role(s) in the process of the vesicle transport via interaction with other vesicle-associated proteins such as p115.     

Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

Oligomerization of mouse alpha 1-syntrophin and self-association of its pleckstrin homology domain 1 containing sequences

Syntrophins are known to self-associate to form oligomers. Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and were found to also oligomerize and in a micromolar Ca(2+)-dependent manner. The oligomerization was localized to the N-terminal pleckstrin homology domain (PH1) or adjacent sequences; the second, C-terminal PH2 domain did not show oligomerization. PH1 was found to self-associate, and calmodulin or Ca(2+)-chelating agents such as ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) could effectively prevent this oligomerization. A single calmodulin bound per syntrophin to cause inhibition of the precipitation. Since calmodulin inhibited syntrophin oligomerization in the presence or absence of Ca(2+), Ca(2+) binding to syntrophin is responsible for the inhibition by EGTA of syntrophin oligomerization.     

Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.     

HSos1 contains a new amino-terminal regulatory motif with specific binding affinity for its pleckstrin homology domain

The protein hSos1 is a Ras guanine nucleotide exchange factor. In the present study, we investigated the function of the amino-terminal region of the hSos1 protein, corresponding to the first 600 residues, which includes the Dbl and pleckstrin homology (DH and PH) domains. We demonstrated, using a series of truncated mutants, that this region is absolutely necessary for hSos1 activity. Our results suggest that the first 200 residues (upstream of DH domain), which we called the HF motif on the basis of their homology with histone H2A, may exert negative control over the functional activity of the whole hSos1 protein. In vitro binding analysis showed that the HF motif is able to interact specifically with the PH domain of hSos1. The amino-terminal region of hSos1 may be associated in vivo with an expressed HF motif. These findings document the existence of the HF motif located upstream of the DH domain in the hSos1 protein. This motif may be responsible for the negative control of hSos1, probably by intramolecular binding with the PH domain.     

The carboxy-terminal pleckstrin homology domain of ROCK interacts with filamin-A

The small GTPase Rho and its effector ROCK/Rho-kinase regulate actin cytoskeletal reorganization through phosphorylation of the regulatory light chain of myosin II. We previously reported that ROCK co-purified with the actin-binding protein filamin-A from HeLa cells. Here, we show that the pleckstrin homology (PH) domain of ROCK, but not the kinase or coiled-coil domain, interacts with filamin-A. We also determined that the PH domain of ROCK binds to the carboxy-terminal region of filamin-A containing the last 24th repeat. ROCK co-localized with filamin-A at the protrusive cell membranes of HeLa cells.     

Ca(2+)/calmodulin directly interacts with the pleckstrin homology domain of AKT1

AKT kinase, also known as protein kinase B, is a key regulator of cell growth, proliferation, and metabolism. The activation of the AKT signaling pathway is one of the most frequent molecular alterations in a wide variety of human cancers. Dickson and coworkers recently observed that Ca(2+).calmodulin (Ca(2+).CaM) may be a common regulator of AKT1 activation (Deb, T. B., Coticchia, C. M., and Dickson, R. B. (2004) J. Biol. Chem. 279, 38903-38911). In our efforts to scan the mRNA-displayed proteome libraries for Ca(2+).CaM-binding proteins, we found that both human and Caenorhabditis elegans AKT1 kinases bound to CaM in a Ca(2+)-dependent manner (Shen, X., Valencia, C. A., Szostak, J., Dong, B., and Liu, R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5969-5974 and Shen, X., Valencia, C. A., Gao, W., Cotten, S. W., Dong, B., Chen, M., and Liu, R. (2007) submitted for publication). Here we demonstrate that Ca(2+).CaM and human AKT1 were efficiently co-immunoprecipitated, and their interaction was direct rather than mediated by other proteins. The binding is in part attributed to the first 42 residues of the pleckstrin homology (PH) domain, a region that is critical for the recognition of its lipid ligands. The PH domain of human AKT1 can disrupt the complex of the full-length AKT1 with Ca(2+).CaM. In addition, Ca(2+).CaM competes with phosphatidylinositol 3,4,5-trisphophate for interaction with the PH domain of human AKT1. Our findings suggest that Ca(2+).CaM is directly involved in regulating the functions of AKT1, presumably by releasing the activated AKT1 from the plasma membrane and/or prohibiting it from re-association with phosphoinositides on plasma membrane.     

Non-canonical interaction of phosphoinositides with pleckstrin homology domains of Tiam1 and ArhGAP9

Pleckstrin homology (PH) domains are phosphoinositide (PI)-binding modules that target proteins to membrane surfaces. Here we define a family of PH domain proteins, including Tiam1 and ArhGAP9, that demonstrates specificity for PI(4,5)P(2), as well as for PI(3,4,5)P(3) and PI(3,4)P(2), the products of PI 3-kinase. These PH domain family members utilize a non-canonical phosphoinositide binding pocket related to that employed by beta-spectrin. Crystal structures of the PH domain of ArhGAP9 in complex with the headgroups of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,5)P(3) reveal how two adjacent phosphate positions in PI(3,4)P(2), PI(4,5)P(2), and PI(3,4,5)P(3) are accommodated through flipped conformations of the bound phospholipid. We validate the non-canonical site of phosphoinositide interaction by showing that binding pocket mutations, which disrupt phosphoinositide binding in vitro, also disrupt membrane localization of Tiam1 in cells. We posit that the diversity in PI interaction modes displayed by PH domains contributes to their versatility of use in biological systems.