Histidine residues 912 and 913 in protein associated with Myc are necessary for the inhibition of adenylyl cyclase activity.
We reported previously that protein associated with Myc (PAM) interacts with the C2 domain of type V adenylyl cyclase (ACV-C2) and that purified PAM is a potent inhibitor of Galphas-stimulated ACV activity (J Biol Chem 276:47583-47589, 2001). The present study was conducted to identify the region in PAM that inhibits ACV activity and to determine whether its binding with the ACV-C2 is necessary and sufficient to inhibit the enzyme. Coexpression of ACV and full-length PAM or its N-terminal third (PAM-N) in COS-7 cells inhibited isoproterenol-stimulated cAMP accumulation. Deletion of the RCC1 homology domains in PAM-N abolished its ability to inhibit isoproterenol-stimulated cAMP formation in cells. Purified GST fusion protein of the second RCC1 homology domain (RHD2) of PAM was sufficient to bind with ACV-C2 and inhibit Galphas-stimulated ACV activity. In addition, deletion of 11 amino acids in GST-RHD2 obliterated its ability to bind with and inhibit ACV. The C terminus of the RHD2 domain bound with ACV-C2 without inhibiting enzyme activity. Furthermore, substitution of His912 and His913 with alanine in the GST-RHD2 obliterated its ability to inhibit ACV without altering binding to ACV-C2. Likewise, H912/913A mutants of both PAM-N and full-length PAM did not inhibit cAMP formation in cells. Thus, the RHD2 domain of PAM is sufficient to inhibit Galphas-stimulated ACV activity and the binding of RHD2 to ACV-C2 is necessary but not sufficient for this inhibition. Moreover, His912 and His913 in PAM are critical for inhibiting ACV.
Department of Pharmacology and Experimental Therapeutics, Loyola University Chicaco, Stritch School of Medicine, 2160 S. First Avenue, Maywood, IL 60153, USA.
Xianlong
X
Gao
Tarun B
TB
Patel
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary; D006367:HeLa Cells; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044767:Ubiquitin-Protein Ligases
D048868:Adaptor Proteins, Signal Transducing; D000067956:Adenylyl Cyclase Inhibitors; D018076:DNA, Complementary; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006639:Histidine; D006899:Mixed Function Oxygenases; C114022:MYCBP2 protein, human; D044767:Ubiquitin-Protein Ligases