Mammalian protein RAP46: an interaction partner and modulator of 70 kDa heat shock proteins

A ubiquitously expressed nuclear receptor-associating protein of approximately 46 kDa (RAP46) was identified recently. Interaction experiments with in vitro-translated proteins and proteins contained in cell extracts revealed that a great variety of cellular regulators associate with RAP46. However, in direct interaction tests by the far-Western technique, only 70 kDa proteins showed up and were identified as members of the 70 kDa heat shock protein (hsp70) family. Interaction is specific since not all members of the hsp70 family bind to RAP46; interaction occurs through their ATP-binding domain. RAP46 forms complexes with hsp70 in mammalian cells and interacts with hsp70 in the yeast two-hybrid system. Consistent with the fact that hsp70 can bind a multitude of proteins, we identified heteromeric complexes of RAP46-hsp70 with some selected proteins, most notably c-Jun. Complex formation is increased significantly by pre-treatment with alkaline phosphatase, thus suggesting modulation of interactions by protein phosphorylation. We observed that RAP46 interferes with efficient refolding of thermally denatured luciferase. Moreover, ATP-dependent binding of misfolded proteins to hsp70 was greatly inhibited by RAP46. These data suggest that RAP46 functions as a regulator of hsp70 in higher eukaryotes.     

Pertussis toxin modification of PC12 cells inhibits a protein phosphatase 2A-like phosphatase

We have found that modification of rat PC12 cells with pertussis toxin resulted in an approximately 50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca2+ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca2+ appears to result from the dephosphorylation of a protein by the Ca2+/calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca2+, the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.     

Autoimmunity to RNA polymerase II is focused at the carboxyl terminal domain of the large subunit

OBJECTIVE: Previous studies have demonstrated antibodies to the large (220 kd) polypeptide subunit of RNA polymerase II (Pol II) in sera from certain patients with scleroderma. In the present study, we sought to identify the autoantigenic region on this polypeptide. METHODS: A recombinant fusion protein, corresponding to the 52-heptapeptide repeat found in the carboxyl terminal domain (CTD) of the large Pol II subunit, was used to identify 15 patient sera that contained autoantibodies. Synthetic peptides CTD7 (representing a single heptapeptide) and CTD18 (representing 2 1/2 heptapeptide repeats) were used in a competitive inhibition assay to define the specificity of these sera and the importance of the CTD as an autoantigen. RESULTS: All 15 sera immunoprecipitated the Pol II subunit from radiolabeled cell extracts, and 11 of them bound the CTD fusion protein in immunoblots. Immunoprecipitation of Pol II was completely inhibited by CTD18 in 5 sera and partially inhibited in 4 additional sera. CONCLUSION: These results indicate that the CTD heptapeptide repeat is a focal point for autoimmune responses in scleroderma. It is likely that the repetitive sequence and high content of charged residues of this structure contribute to its role as an autoantigen.     

Direct interaction of the KRAB/Cys2-His2 zinc finger protein ZNF74 with a hyperphosphorylated form of the RNA polymerase II largest subunit

We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys2-His2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with pre- mRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.     

The heat shock response inhibits RANTES gene expression in cultured human lung epithelium

The chemokine RANTES is thought to be involved in the pathophysiology of inflammation-associated acute lung injury. Although much is known regarding signals that induce RANTES gene expression, relatively few data exist regarding signals that inhibit RANTES gene expression. The heat shock response, a highly conserved cellular defense mechanism, has been demonstrated to inhibit a variety of lung proinflammatory responses. We tested the hypothesis that induction of the heat shock response inhibits RANTES gene expression. Treatment of A549 cells with TNF-alpha induced RANTES gene expression in a concentration-dependent manner. Induction of the heat shock response inhibited subsequent TNF-alpha-mediated RANTES mRNA expression and secretion of immunoreactive RANTES. Transient transfection assays involving a RANTES promoter-luciferase reporter plasmid demonstrated that the heat shock response inhibited TNF-alpha-mediated activation of the RANTES promoter. Inhibition of NF-kappaB nuclear translocation with isohelenin inhibited TNF-alpha-mediated RANTES mRNA expression, indicating that RANTES gene expression is NF-kappaB dependent in A549 cells. Induction of the heat shock response inhibited degradation of the NF-kappaB inhibitory protein, I-kappaBalpha but did not significantly inhibit phosphorylation of I-kappaBalpha. We conclude that the heat shock response inhibits RANTES gene expression by a mechanism involving inhibition of NF-kappaB nuclear translocation and subsequent inhibition of RANTES promoter activation. The mechanism by which the heat shock response inhibits NF-kappaB nuclear translocation involves stabilization of I-kappaBalpha, without significantly affecting phosphorylation of I-kappaBalpha.