Heteroprotein Complex Research Articles

Inositol 1,4,5-triphosphate-associated cGMP kinase substrate: Basis Sequence: Mouse.

Inositol 1, 4, 5-trisphosphate (IP3) receptor associated cyclic GMP (cGMP) kinase substrate (IRAG, also known as Mrv1) is a type-2 integral membrane endoplasmic reticulum (ER) protein, which interacts with IP3 Receptor type 1 (IP3R1), cGMP kinase I-β (cGKI β) and other associated proteins. It plays a key role in NO, cGMP, and cGKI β mediated inhibition of IP3R1 activity and thus relaxes smooth muscle tone and inhibits platelet aggregation. As a scaffolding protein Mrv1 maintains the conformation of a heteroprotein complex containing cGKI β, IP3R1 and other proteins and enables efficient activity of cGKI β within the complex. Increased expression of Mrv1 or IRAG in the absence of tumor related transcription factor in pancreatic cancer cells suggest that it might be involved in tumorigenesis. Downregulation of Mrv1 during megakaryocyte maturation indicates that it is involved in cell growth and differentiation.

Protein Kinase C Phosphorylation Disrupts Na+/H+ Exchanger Regulatory Factor 1 Autoinhibition and Promotes Cystic Fibrosis Transmembrane Conductance Regulator Macromolecular Assembly

An emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function and cross-talk. In this study, we determined how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na(+)/H(+) exchanger regulatory factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls fluid and electrolyte transport across cell membranes. NHERF directs polarized expression of receptors and ion transport proteins in epithelial cells, as well as organizes the homo- and hetero-association of these cell surface proteins. NHERF contains two modular PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain. Previous studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We show that PKC phosphorylates two amino acid residues Ser-339 and Ser-340 in the C-terminal domain of NHERF, but a serine 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/S340D of NHERF has increased affinity and stoichiometry when binding to C-CFTR. Moreover, solution small angle x-ray scattering indicates that the PDZ2 and C-terminal domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant indicating that PKC phosphorylation disrupts the autoinhibition interactions in NHERF. The results demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and thus controls the stoichiometry of NHERF to assemble protein complexes.

Functional dissection of Ctr4 and Ctr5 amino-terminal regions reveals motifs with redundant roles in copper transport

Copper uptake in the fission yeast Schizosaccharomyces pombe is carried out by a heteromeric complex formed by two proteins, Ctr4 and Ctr5. In this study, a stable expression system using integrative plasmids was developed to investigate the respective roles of Ctr4 and Ctr5 in copper transport. It was shown that expression of full-length Ctr4 or truncated Ctr4 containing residues 106-289 was required for localization of Ctr5 to the plasma membrane. Likewise, when the full-length Ctr5 or truncated Ctr5 from residues 44-173 was co-expressed with Ctr4, this protein was visualized at the periphery of the cell. To determine the importance of the Mets motifs (consisting of five methionines arranged as Met-X2-Met-X-Met, where X is any amino acid) of Ctr4 and Ctr5 in the heteroprotein complex, we co-expressed Ctr5 lacking the Mets motif and Cys-X-Met-X-Met sequence with wild-type Ctr4 or its mutant derivatives. Conversely, Ctr4 lacking the Mets motif and Met(122) was expressed with wild-type Ctr5 or its mutant derivatives. These experiments revealed that the five Mets motifs of Ctr4 and the Ctr4 residue Met(122) have equally important roles in copper assimilation. Furthermore, the two partially overlapping Mets motifs and the Cys-X-Met-X-Met sequence in Ctr5 have redundant functions in copper transport, with the latter sequence making a greater contribution than the former. Together, the data reveal that co-expression of both Ctr4 and Ctr5 is necessary for the proper function and localization of the heteroprotein complex to the plasma membrane. Once on the cell surface, the N-terminal regions of Ctr4 and Ctr5 can function independently to transport copper; however, the greatest efficiency is achieved when both N termini are present.

Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1

UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulfite to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher affinity for the substrate sulfite than the recombinant SQD1 protein itself. The SQD1 protein co-purified with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxin-dependent glutamate synthase (FdGOGAT). While the first two proteins are known to interact with many other proteins, the identification of FdGOGAT was most intriguing because this 160 kDa protein contains an FMN cofactor known to bind sulfite in vitro. Using different constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for specific binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulfite to SQD1.

The physical association of multiple molecular chaperone proteins with mutant p53 is altered by geldanamycin, an hsp90-binding agent.

Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin-proteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted that many transforming p53 mutants are found in stable physical association with molecular chaperones of the hsp70 class. To explore a possible role for aberrant chaperone interactions in mediating the altered function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a temperature-sensitive murine p53 mutant. In lysate prepared from A1-5 cells grown under mutant temperature conditions, hsp70 coprecipitated with p53Val135 as previously reported by others, but in addition, other well-recognized elements of the cellular chaperone machinery, including hsp90, cyclophilin 40, and p23, were detected. Under temperature conditions favoring wild-type p53 conformation, the coprecipitation of chaperone proteins with p53 was lost in conjunction with the restoration of its transcriptional activating activity. Chaperone interactions similar to those demonstrated in A1-5 cells under mutant conditions were also detected in human breast cancer cells expressing two different hot-spot mutations. To examine the effect of directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been shown to alter the chaperone associations regulating the function of unliganded steroid receptors. GA treatment of cells altered heteroprotein complex formation with several different mutant p53 species. It increased p53 turnover and resulted in nuclear translocation of the protein in A1-5 cells. GA did not, however, appear to restore wild-type transcriptional activating activity to mutant p53 proteins in either A1-5 cells or human breast cancer cell lines.

Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells.

When isolated from cells grown under hormone-free conditions, the glucocorticoid receptor (GR) is known to exist as a large heteroprotein complex that contains, among its multiple components, the stress protein hsp90 (heat shock protein 90). To explore hsp90's role in mediating glucocorticoid hormone action, we have examined the effects of a selective hsp90-binding agent, geldanamycin (GA), or GR structure and function. Using a steroid-responsive reporter construct, we found that GA inhibited the dexamethasone-dependent transactivating activity of GR in transfected cells. At the molecular level, GA bound hsp90, but not GR, in a stable and specific manner in intact cells. GA treatment of cells did not inhibit coprecipitation of hsp90 or hsp70 with the GR but did result in a complete loss of the recently described p23 protein from GR immunoprecipitates. This drug-induced alteration in GR heteroprotein complex composition was associated with a rapid (15-30 min), noncompetitive loss of dexamethasone-binding activity. Longer exposures of cells to GA (2-8 h), resulted in a marked decline in the cellular level of GR protein. Pulse-chase data revealed that this decline resulted from a decrease in GR protein stability, not rate of synthesis. GA-induced declines in GR protein level were blocked by cotreatment of cells with lactacystin, a selective inhibitor of 20S proteasome activity, suggesting the possible involvement of the ubiquitin-proteasome pathway in mediating GA-induced decreases in GR protein abundance. Overall, these findings provide direct pharmacological evidence that hsp90 function is required to maintain both the hormone-binding activity and stability of the GR protein in intact cells and suggest that hsp90 function may provide a novel target for the modulation of steroid hormone signaling.

Possible Role for Serine/Threonine Phosphorylation in the Regulation of the Heteroprotein Complex between the hsp90 Stress Protein and the pp60v-src Tyrosine Kinase

The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-src chaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90. Although phosphorylation of the total pool of immunoprecipitable pp60v-src was unchanged, okadaic acid slightly increased phosphoserine and phosphothreonine levels specifically in pp60v-src bound to heat-shock protein 90. The low level of tyrosine phosphorylation in the pp60v-src complexed with heat-shock protein 90 was further decreased by okadaic acid. Interestingly, okadaic acid-stabilized hyperphosphorylation of the heat-shock protein 90-pp60v-src complex lowered the level of pp60v-src in cell membranes, the functional location for pp60v-src. We suggest that serine/threonine phosphorylation of heat-shock protein 90 and/or pp60v-src functions as a regulatory molecular trigger to release pp60v-src from the chaperone complex at the inner surface of cell membranes.

Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.

The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity. To identify mechanisms other than kinase inhibition for drug-mediated reversion, we prepared a solid phase-immobilized geldanamycin derivative and affinity precipitated the molecular targets with which the drug interacted. In a range of cell lines, immobilized geldanamycin bound elements of a major class of heat shock protein (HSP90) in a stable and pharmacologically specific manner. Consistent with these binding data, we found that soluble geldanamycin and herbimycin A inhibited specifically the formation of a previously described src-HSP90 heteroprotein complex. A related benzoquinone ansamycin that failed to revert transformed cells did not inhibit the formation of this complex. These results demonstrate that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.

Reversion of the Maple Syrup Urine Disease Phenotype of Impaired Branched Chain α-Ketoacid Dehydrogenase Complex Activity in Fibroblasts from an Affected Child

Branched chain alpha-ketoacid dehydrogenase is a heteroprotein complex of mitochondria and commits the branched chain alpha-ketoacids to their catabolic fate. Inherited nuclear mutations in humans decrease the activity of this complex and result in maple syrup urine disease. Here we demonstrate the restoration of branched chain alpha-ketoacid dehydrogenase activity to fibroblasts from a child with this disorder by transfection with a cDNA for the prebranched chain acyltransferase. Prior to transfection these fibroblasts contained the prebranched chain acyltransferase gene but failed to transcribe the gene and thus lacked the protein. Regulation of the restored complex by phosphorylation mechanisms resembles that of wild-type cells. These results describe a human cell modeling system for testing engineered proteins and support the possibility of gene replacement therapy for this human disorder.