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.
Zeiner
M
M
Institut für Biologische Chemie, Universität Heidelberg, Im Neuenheimer Feld 501, D-69120 Heidelberg, Germany.
Gebauer
M
M
Gehring
U
U
D000469:Alkaline Phosphatase; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D008322:Mammals; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010763:Phosphorylase b / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011489:Protein Denaturation; D017510:Protein Folding; D016755:Proto-Oncogene Proteins c-jun / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D018411:Spodoptera; D014157:Transcription Factors; D014162:Transfection
C091732:BCL2-associated athanogene 1 protein; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D008156:Luciferases; D010763:Phosphorylase b; D000469:Alkaline Phosphatase; C063842:dnaK protein, E coli