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PubMed2001

The membrane-associated guanylate kinase protein MAGI-1 binds megalin and is present in glomerular podocytes.

The transmembrane endocytic receptor glycoprotein 330/megalin (hereafter referred to as megalin) is localized to the apical membrane domain of epithelial cells, where it is involved in the uptake of proteins from extracellular sources. The cytoplasmic domain of megalin contains amino acid motifs that have the potential to bind to other proteins, which may influence its localization or function. The yeast two-hybrid system was used to search for proteins that bind to the cytoplasmic tail of megalin, and a protein fragment from a mouse embryonic cDNA library that contained a single PDZ domain was identified. This protein, which was named glycoprotein 330-associated protein (GASP), appears to be a truncated mouse counterpart of the human and rat proteins atrophin-1-interacting protein-1 and synaptic scaffolding molecule, respectively. The interaction of GASP with megalin is mediated by the PDZ domain of GASP binding to the DSDV motif found at the carboxyl-terminus of megalin. A mutant version of megalin that lacks the terminal valine is unable to bind to GASP, illustrating the PDZ domain-dependent interaction between these two proteins. A close homolog of GASP, i.e., membrane-associated guanylate kinase with inverted orientation-1 (MAGI-1), is more ubiquitous in its tissue distribution (including kidney) and is also able to specifically bind to megalin via its fifth PDZ domain. Immunofluorescence studies of adult kidney revealed that MAGI-1 is expressed in the glomerulus of the kidney, in a manner that parallels the expression of the podocyte-specific protein glomerular epithelial protein 1. Western analysis of endogenous MAGI-1 from glomerular preparations suggests that it is associated with the cytoskeleton and seems to be expressed in a different form, compared with cell line-derived endogenous MAGI-1. The association of megalin with MAGI-1 may allow the assembly of a multiprotein complex, in which megalin may serve a nonendocytic function in glomerular podocytes.

Amino Acid Sequence↗

PubMed2001

Polycystin: new aspects of structure, function, and regulation.

Polycystin-1 is a modular membrane protein with a long extracellular N-terminal portion that bears several ligand-binding domains, 11 transmembrane domains, and a > or =200 amino acid intracellular C-terminal portion with several phosphorylation signaling sites. Polycystin-1 is highly expressed in the basal membranes of ureteric bud epithelia during early development of the metanephric kidney, and disruption of the PKD1 gene in mice leads to cystic kidneys and embryonic or perinatal death. It is proposed that polycystin-1 functions as a matrix receptor to link the extracellular matrix to the actin cytoskeleton via focal adhesion proteins. Co-localization, co-sedimentation, and co-immunoprecipitation studies show that polycystin-1 forms multiprotein complexes with alpha2beta1-integrin, talin, vinculin, paxillin, p130cas, focal adhesion kinase, and c-src in normal human fetal collecting tubules and sub-confluent epithelial cultures. In normal adult kidneys and confluent epithelial cultures, polycystin-1 is downregulated and forms complexes with the cell-cell adherens junction proteins E-cadherin and beta-, gamma-, and alpha-catenin. Polycystin-1 activation at the cell membrane leads to intracellular signaling via phosphorylation through the c-Jun terminal kinase and wnt pathways leading to activation of AP-1 and TCF/LEF-dependent genes, respectively. The C-terminal of polcystin-1 has been shown to be phosphorylated by c-src at Y4237, by protein kinase A at S4252, and by focal adhesion kinase and protein kinase X at yet-to-be identified residues. Inhibition of tyrosine phosphorylation or increased cellular calcium increases polycystin-1 focal adhesion complexes versus polycystin-1 adherens junction complexes, whereas disruption of the actin cytoskeleton dissociates all polycystin-1 complexes. Genetic evidence suggests that PKD1, PKD2, NPHP1, and tensin are in the same pathway.

Animals↗

PubMed2003

The peripheral benzodiazepine receptor: a promising therapeutic drug target.

The peripheral benzodiazepine receptor (PBR) is a critical component of the mitochondrial permeability transition pore (MPTP), a multiprotein complex located at the contact site between inner and outer mitochondrial membranes, which is intimately involved in the initiation and regulation of apoptosis. PBR is a small evolutionary conserved protein, located at the surface of the mitochondria where it is physically associated with the voltage-dependent anion channel (VDAC) and adenosine nucleotide translocase (ANT) that form the backbone of MPTP. PBR is widely distributed throughout the body and has been associated with numerous biological functions. Consistent with its localization in the MPTP, PBR is involved in the regulation of apoptosis, but also in the regulation of cell proliferation, stimulation of steroidogenesis, immunomodulation, porphyrin transport, heme biosynthesis, anion transport and regulation of mitochondrial functions. The recent literature on PBR is reviewed here. Specifically, we highlight numerous results suggesting that the use of specific PBR ligands to modulate PBR activity may have potential therapeutic applications and might be of significant clinical benefit in the management of a large spectrum of different indications including cancer, auto-immune, infectious and neurodegenerative diseases. In addition, we present the proposed mechanisms by which the molecules exerted these effects, particularly oriented on the modulation of the MPTP activities.

Amino Acid Sequence↗

PubMed2004

Ubiquitin-protein ligases--novel therapeutic targets?

Intracellular protein degradation is a tightly regulated process that in many cases is controlled by protein ubiquitylation. The ubiquitin pathway is a major route by which cells not only remove normal proteins at the appropriate time but also abnormally folded normal or mutant, cytoplasmic and membrane, proteins. This has led to a major impetus to identify constituents of the pathway. The key components that regulate substrate ubiquitylation are the ubiquitin-protein ligases. Ligases come in many forms, from single proteins to very large multiprotein complexes. Specificity of targeting can be modulated by the requirement for post-translational modifications such as phosphorylation, hydroxylation or oxidation of the substrate and, in some cases, the ligase itself. The requirement for substrate modification prior to ubiquitylation allows the same ligase to target different substrates within the same cell at different times. Abnormal intracellular protein processing is a common feature of many human diseases including neurodegenerative diseases and cancer. It may not represent the causative factor that initiates the disease process but may be a downstream regulator of the toxic effect. These abnormalities often arise from the loss of a key protein-protein interaction. As a consequence, mutated proteins can have very different half-lives from their normal counterparts. This can affect the levels of their activity and/or lead to the formation of protein aggregates (inclusion bodies/aggresomes). In this review, we aim to highlight examples of diseases where abnormal protein ubiquitylation is proposed to be a key regulator of the disease process. The recent success of the proteasome inhibitor Bortezomib (PS-341) for treatment of relapsed, refractory myeloma suggests that the modulation of individual ubiquitin-protein ligase activities with synthetic agents may represent a novel approach that has enormous potential for the treatment of a wide range of diseases.

Cysteine Endopeptidases↗

PubMed2003

Resistance to apoptosis: mechanism for the development of HIV reservoirs.

New insights into the physiological cell death program in mammalian cells have further confounded the central issue of T lymphocyte destruction during HIV infection. Although infected and uninfected cells die following infection, recent evidence indicates that infected and uninfected cells may have unique pathways controlling death. Two widely accepted models for apoptosis have been described, namely the intrinsic and extrinsic apoptotic pathway. In the extrinsic pathway, ligation of TNF death ligands to their receptors causes oligomerization of the death receptors and recruitment of adaptor proteins typically involving caspase 8 activation. In the intrinsic pathway, apoptotic signals converge on mitochondria to cause activation and subsequent release of cytochrome c, which leads to formation of a multiprotein complex containing Apaf-1, cytochrome c, dATP and procaspase 9. Expression of HIV proteins alters these pathways resulting in enhanced levels of apoptosis. Although HIV infection results in T cell apoptosis, under some circumstances a small fraction of CD4+ T cells and macrophages do not die following infection, indicating that this may be a critical step in the development of viral reservoirs. In addition, monocyte differentiation into macrophages leads to an apoptosis resistant phenotype characterized by upregulation of antiapoptotic molecules and lower levels of proapoptotic molecules. The development of these stable antiretroviral resistant cells represents the major obstacle in achieving a complete sterile cure. However, this provides a unique opportunity to further understand the regulation of apoptosis and may facilitate novel immune based therapies aimed at modifying apoptosis in HIV disease.

Apoptosis↗

PubMed1992

Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review).

NF-kappa B is a multiprotein complex that can activate a great variety of genes involved in early defence reactions of higher organisms. In nonstimulated cells, NF-kappa B resides in the cytoplasm in an inactive complex with the inhibitor I kappa B. Pathogenic stimuli cause release of I kappa B and allow NF-kappa B to enter the nucleus, bind to DNA control elements and, thereby, induce the synthesis of mRNA. A puzzling feature of NF-kappa B is that its activation is triggered by a great variety of agents. These include the cytokines interleukin-1 and tumor necrosis factor, viruses, double-stranded RNA, endotoxins, phorbol esters, UV light and ionizing radiation. We recently found that also low concentrations of H2O2 activate NF-kappa B and that various antioxidants prevent the induction by H2O2. Subsequent analysis revealed that antioxidants not only suppress the activation of NF-kappa B by H2O2 but by all other inducers tested so far. In this review, we will discuss the evidences that NF-kappa B is an oxidative stress-responsive transcription factor of higher eukaryotic cells.

Animals↗

PubMed2001

Secretases as therapeutic targets for the treatment of Alzheimer's disease.

The extracellular deposition of short amyloid peptides in the brain of patients is thought to be a central event in the pathogenesis of Alzheimer's Disease. The generation of the amyloid peptide occurs via a regulated cascade of cleavage events in its precursor protein, A beta PP. At least three enzymes are responsible for A beta PP proteolysis and have been tentatively named alpha-, beta- and gamma-secretases. The recent identification of several of these secretases is a major leap in the understanding how these secretases regulate amyloid peptide formation. Members of the ADAM family of metalloproteases are involved in the non-amyloidogenic alpha-secretase pathway. The amyloidogenic counterpart pathway is initiated by the recently cloned novel aspartate protease named BACE. The available data are conclusive and crown BACE as the long-sought beta-secretase. This enzyme is a prime candidate drug target for the development of therapy aiming to lower the amyloid burden in the disease. Finally, the gamma-secretases are intimately linked to the function of the presenilins. These multi-transmembrane domain proteins remain intriguing study objects. The hypothesis that the presenilins constitute a complete novel type of protease family, and are cleaving A beta PP within the transmembrane region, remains an issue of debate. Several questions remain unanswered and direct proof that they exert catalytic activity is still lacking. The subcellular localization of presenilins in neurons, their integration in functional multiprotein complexes and the recent identification of additional modulators of gamma-secretase, like nicastrin, indicate already that several players are involved. Nevertheless, the rapidly increasing knowledge in this area is already paving the road towards selective inhibitors of this secretase as well. It is hoped that such drugs, possibly in concert with the experimental vaccination therapies that are currently tested, will lead to a cure of this inexorable disease.

Alzheimer Disease↗

PubMed1997

E-cadherin engagement stimulates tyrosine phosphorylation.

Cadherins are cell adhesion molecules concentrated at intercellular adherens junctions, where they form a multiprotein complex with cytoplasmic catenins. Although cell-cell interactions affect many aspects of cell behavior, little is known about signaling pathways triggered by cadherin engagement. We show here that E-cadherin-mediated cell-cell adhesion leads to a rapid increase in tyrosine phosphorylation at sites of cell-cell contact and that this stimulation of tyrosine phosphorylation can be mimicked by aggregation of E-cadherin with antibodies. The proteins that become phosphorylated are distinct from those previously shown to be tyrosine phosphorylated in response to integrin-mediated adhesion and include ras-GAP. We also find that E-cadherin-mediated tyrosine phosphorylation is not required for the assembly of adherens-type junctions.

Cadherins↗

PubMed2001

Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst.

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47(phox), a major cytosolic component of this oxidase. Protein kinase C zeta (PKC zeta), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC zeta in p47(phox) phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47(phox) with recombinant PKC zeta induced a time- and concentration-dependent phosphorylation of p47(phox) with an apparent K(m) value of 2 microM. Phosphopeptide mapping analysis of p47(phox) showed that PKC zeta phosphorylated fewer selective sites in comparison to "conventional" PKCs. Serine 303/304 and serine 315 were identified as targets of PKC zeta by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC zeta that correlated to that of p47(phox). A cell-permeant-specific peptide antagonist of PKC zeta inhibited both fMLP-induced phosphorylation of p47(phox) and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC(50) of 10 microM), but not that induced by PMA. The inhibition of PKC zeta expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 microM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47(phox) is a substrate for PKC zeta and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.

Amino Acid Sequence↗

PubMed2004

Distinct sequences in the cytoplasmic domain of complement receptor 2 are involved in antigen internalization and presentation.

B cells express randomly rearranged surface Ig that forms part of a multiprotein complex known as the B cell receptor (BCR). Recognition of Ag via this receptor results in its capture, internalization, proteolysis and presentation to CD4+ T cells. The recognition of Ag by CD4+ T cells is critical for the selection of individual B cells, leading to the eventual secretion of a high affinity version of the BCR as an effective circulating Ab. B cells also express other receptors that recognize Ags associated with components of innate immunity. One of these receptors, CR2, binds Ags coated with activated complement components. Studies have shown that cross-linking CR2 and the BCR with complement-tagged Ags leads to enhanced Ag presentation by B cells. In addition, Ags targeted to B cell CR2 in the absence of BCR coligation are also efficiently presented to T cells. In this report, we identify several distinct sequences within the cytoplasmic domain of mouse CR2 (mCR2) that are essential for mCR2-mediated Ag presentation in both the presence and the absence of BCR cross-linking. The finding that distinct sequences in the cytoplasmic domain of mCR2 are essential for BCR-independent Ag presentation leads us to propose a novel role for CR2.

Amino Acid Sequence↗

PubMed2004

Geranylgeraniol regulates negatively caspase-1 autoprocessing: implication in the Th1 response against Mycobacterium tuberculosis.

Caspase-1 is a cysteine protease composed by two 20-kDa and two 10-kDa subunits that processes pro-IL-1beta and pro-IL-18 to their mature forms. This enzyme is present in cells as a latent zymogen that becomes active through a tightly regulated proteolytic cascade. Activation is initiated by the oligomerization of an adaptor molecule, or by the formation of a multiprotein complex named inflammasome. Negative regulation of caspase-1 activation is exerted by proteins that compete with the adaptor molecule or with the inflammasome formation. We previously reported that fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, increases caspase-1 activity in PBMC. This effect was strengthened by Mycobacterium tuberculosis, rending an exacerbated IL-1beta, IL-18, and IFN-gamma production. Mevalonate, the product of 3-hydroxy-3-methylglutaryl coenzyme A reductase, is a precursor for both nonsterol isoprenoid and sterol formation. In this study, we studied the involvement of mevalonate derivatives in the regulation of caspase-1 activation. Inhibition of sterol formation by SKF-104976 or haloperidol had no effect on IL-1beta release. However, the isoprenoid geranylgeraniol prevented both caspase-1 activation and the exacerbated IL production induced by fluvastatin. This isoprenoid significantly reduced the release of IL-18 and IFN-gamma by PBMC treated with mycobacteria, even in the absence of fluvastatin. In correlation with the increased caspase-1 activity, fluvastatin stimulated the proforms cleavage, enhancing the formation of active subunit p10. Geranylgeraniol not only prevented this effect, but induced proforms accumulation. Present results suggest that, once the proteolytic cascade is initiated, geranylgeraniol may exert an additional negative regulation on caspase-1 cleavage process.

Caspase 1↗

PubMed2005

Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development.

It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.

Adaptor Proteins, Signal Transducing↗

PubMed2007

Analysis of apoptosome dysregulation in pancreatic cancer and of its role in chemoresistance.

The apoptosome is a multiprotein complex mediating the mitochondrial pathway of cell death. Its importance during development has been clearly demonstrated by knocking out key genes in mouse. APAF1 is the core protein of the apoptosome and its dosage is also critical in various cancer types, i.e., melanoma, germ line tumor, gastrointestinal cancer and B-type chronic lymphocytic leukemia. This is generally due to inactivation of the APAF1 locus by epigenetic phenomena or by activity of promoter regulators. We investigated the putative roles of the apoptosome in pancreatic ductal adenocarcinoma (PDAC). We found that both APAF1 mRNA and protein are dysregulated in human PDAC samples. Similarly, several PDAC cell lines exhibited variable levels of both APAF1 protein and mRNA. The response to cell death induction and its biochemical features were assessed by treatment of each line with commonly used chemotherapeutic agents. We found that the apoptosome pathway was not functional in most cell lines upon cytochrome c release from mitochondria. In addition, we restored APAF1 and Caspase-9 dosage in Panc-1 cells, where the apoptosome is downregulated, by overexpressing the murine cDNA of the two molecules, and we improved the death response to chemotherapeutic agents.

Animals↗

PubMed2005

Gam1 and the SUMO pathway.

Post-translational modifications of proteins have critical roles in many cellular processes because they can cause rapid changes in the functions of preexisting proteins, multiprotein complexes and subcellular structures. Sumoylation, a ubiquitin-like dynamic and reversible post-translational modification system, is an enzymatic cascade leading to the covalent attachment of SUMO to it target proteins. This modification involves three steps and different enzymes: SUMO-activating enzyme E1 (SAE1/SAE2), SUMO-conjugating enzyme E2 (UBC9), SUMO ligases E3s, and SUMO cleaving enzymes. Although the identification of SUMO-modified substrates has progressed rapidly, the biological function of SUMO and regulation of SUMO conjugation are still not well understood. Some viral proteins have been identified as substrates for SUMO modification as well as altering the sumoylation status of host cell proteins. We have been studying an unusual adenoviral protein, Gam1, a strong and global transcriptional activator of both viral and cellular genes that inactivates HDAC1. We have recently expanded the known functions of Gam1 by demonstrating that Gam1 also inhibits the SUMO pathway by interfering with the activity of E1 heterodimer (SAE1/SAE2), leading to the accumulation of SUMO-unmodified substrates. Our data provides a clear example of the effects of a viral infection on host sumoylation and supports the idea that viruses have multifunctional protein that can target essential biochemical pathways.

Adenosine Triphosphatases↗

PubMed2006

Phospho-dependent protein recognition motifs contained in C/EBP family of transcription factors: in silico studies.

CCAAT/enhancer-binding proteins (C/EBPs) are transcriptional regulators implicated in cell proliferation, differentiation, survival, and tumorigenesis. Their biological activities require interactions with several protein partners. This report presents insights from in silico analysis aimed at identifying phosphorylation-dependent protein recognition motifs in C/EBPs. (1) All C/EBP variants contain intrinsically disordered Ser/Thr- and Pro-rich segments with potential docking sites for WW and Polo-box domains of prolyl isomerase Pin1 and Polo-like kinases (Plks), respectively. (2) Consensus phosphorylation sequences for Plks are located in a highly conserved region of transactivation domains, suggesting that Plks might modulate transcriptional activities of C/EBPs in a cell cycle-dependent manner. (3) Phosphorylation at these positions, as well as at conserved Ser in the extended basic region, would create phosphoserine-containing motifs (pSXXF/Y/I/L), which could be recognized by BRCT repeats containing proteins such as the PAX-transactivation-domain-interacting protein (PTIP), and the breast cancer-associated protein (BRCA1). Proteins containing BRCT domains serve as scaffolds, mediating protein-protein interactions and formation of functional multiprotein complexes involved in DNA repair and cell cycle control. These findings add a new perspective to studies aimed at elucidation of molecular mechanisms underlying the diverse functions of C/EBPs.

Amino Acid Motifs↗

PubMed1999

DNA-dependent protein kinase and related proteins.

The DNA-dependent protein kinase (DNA-PK) is a nuclear protein serine/threonine kinase that must bind to DNA double-strand breaks to be active. We and others have shown that it is a multiprotein complex comprising an approx. 465 kDa catalytic subunit (DNA-PKcs) and a DNA-binding component, Ku. Notably, cells defective in DNA-PK are hypersensitive to ionizing radiation. Thus X-ray-sensitive hamster xrs-6 cells are mutated in Ku, and rodent V3 cells and cells of the severe combined immune-deficient (Scid) mouse lack a functional DNA-PKcs. Cloning of the DNA-PKcs cDNA revealed that it falls into the phosphatidylinositol (PI) 3-kinase family of proteins. However, biochemical assays indicate that DNA-PK contains no intrinsic lipid kinase activity, but is instead a serine/threonine kinase. We have also found that DNA-PK activity can be inhibited by the PI 3-kinase inhibitors wortmannin and LY294002. Consistent with its proposed role in genome surveillance and the detection of DNA damage, DNA-PKcs is most similar to a subset of proteins involved in cell-cycle checkpoint control and signalling of DNA damage. Furthermore, the recent cloning of the gene mutated in ataxia-telangiectasia (A-T) patients, named ATM (A-T mutated), has revealed that the product of this gene is also a PI 3-kinase family member and is related to DNA-PKcs. Although much is known about the clinical symptoms and cellular phenotypes that arise from disruption of the A-T gene, little is known about the biochemical action of ATM in response to DNA damage. Given its sequence similarity with DNA-PKcs, we speculate that ATM may function in a manner similar to DNA-PK.

Androstadienes↗

PubMed1999

Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism.

Animals↗

PubMed1999

An inositolphosphate-binding immunophilin, IPBP12.

A novel inositolphosphate-binding protein has been identified and shown to be an immunophilin. This protein, which was isolated from human erythrocyte membranes and from K562 (human erythroleukemia) cell membranes, has robust peptidylprolyl cis-trans isomerase activity that is strongly inhibited by nanomolar concentrations of FK506 or rapamycin, indicating a member of the FKBP (FK506-binding protein) class. However, unlike the cytosolic FKBP12, the isomerase activity of this membrane-associated immunophilin is strongly inhibited by nanomolar concentrations of inositol 1,4, 5-trisphosphate (IP(3)), inositol 1,3,4,5-tetrakisphosphate (IP(4)), and phosphatidylinositol 4- and 4,5-phosphates, which are suggested to be physiological ligands. The demonstration of a single 12-kD protein that binds both IP(4) or IP(3) and anti-FKBP12 provides strong support for the inositolphosphate-binding immunophilin having an apparent mass of 12 kD, and it is suggested that the protein might be called IPBP12 for 12-kD inositol phosphate binding protein. When an internal tryptic peptide derived from IPBP12 was sequenced, a sequence also present in human cytokeratin 10 was identified, suggesting a cytoskeletal localization for the immunophilin. While purifying IPBP12, it was found that it is immunoprecipitated with specific proteins that include a protein kinase and a phosphoprotein phosphatase. The latter is indicated to be phosphoprotein phosphatase 2A (PP-2A). It is suggested that immunophilins promote the assembly of multiprotein complexes that often include a protein kinase or a phosphoprotein phosphatase or both.

Amino Acid Sequence↗