Activity-dependent interaction of the intracellular domain of rat trkA with intermediate filament proteins, the beta-6 proteasomal subunit, Ras-GRF1, and the p162 subunit of eIF3.
Many responses to nerve growth factor (NGF) are regulated through the receptor tyrosine kinase trkA. To understand more fully the functions of trkA in NGF responsive cells, we have expressed the intracellular domain of rat trkA as a fusion protein with the yeast gal4 transcription factor, and used the fusion protein to probe rat and mouse cDNA libraries by the yeast two-hybrid system. We have identified a direct interaction between the intracellular domain of trkA and two members of the intermediate filament (IF) family of proteins, the guanine-nucleotide exchange protein Ras-GRF1, the p162 subunit of eIF3, and the beta-6 proteasome subunit. The interactions are dependent on an active trkA kinase, and RasGRF1, the beta-6 proteasomal subunit, and peripherin are directly phosphorylated by trkA. The interaction with trkA is not affected by mutations at either Tyr499 or Tyr794, the two major phosphotyrosine residues essential to the activation and receptor binding of Shc, FRS-2/SNT, and phospholipase Cgamma-1, and it is highly specific in vitro for trkA, with little or no binding observed with trkB and/or trkC. The results show that trkA may play a regulatory role in a variety of cellular functions in addition to neuritogenesis, including regulated protein degradation and transcriptional activation.
MacDonald
J I
JI
The John P. Robarts Research Institute, Neurodegeneration Group, London, Ontario, Canada.
Verdi
J M
JM
Meakin
S O
SO
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020932:Nerve Growth Factor / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D051381:Rats; D020917:Receptor, trkA / Q000235:genetics* / Q000378:metabolism*; D020813:Receptor, trkB / Q000378:metabolism; D020812:Receptor, trkC / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism; D020666:ras-GRF1 / Q000378:metabolism*
D004268:DNA-Binding Proteins; C105529:ELF3 protein, human; D007381:Intermediate Filament Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; C108663:REG proteasome activator subunit beta; D014157:Transcription Factors; D020666:ras-GRF1; D014443:Tyrosine; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D020813:Receptor, trkB; D020812:Receptor, trkC