A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis.
Mutations in the tuberous sclerosis 2 (TSC2) gene product have been genetically linked to the pathology of both tuberous sclerosis (TSC) and the gender-specific lung disease, lymphangioleiomyomatosis (LAM). Both diseases are classified as disorders of cellular migration, proliferation, and differentiation. Earlier studies from our laboratory (1) linked TSC2 with steroid/nuclear receptor signaling. Studies presented here provide evidence for calmodulin (CaM) signaling in the propagation of this TSC2 activity. Far Western screening of a lambda phage human brain cDNA library to identify interacting proteins for the TSC2 gene product (tuberin) yielded multiple clones encoding human CaM. Direct binding with 32P-labeled tuberin demonstrated Ca2+-dependent binding to CaM-Sepharose which was lost upon deletion of the C-terminal 72 residues. The sequence (1740)WIARLRHIKRLRQRIC(1755) was identified as one capable of forming a basic amphipathic helix indicative of CaM binding domains in known calmodulin binding proteins. Studies with a synthetic peptide of this sequence demonstrated very tight Ca2+-dependent binding to CaM as judged by tryptophan fluorescence perturbation studies and phosphodiesterase activation by CaM. Deletion mutagenesis studies further suggested that this CaM binding domain is required for tuberin modulation of steroid receptor function and that mutations in this region may be involved in the pathology of TSC and LAM.
Noonan
Daniel J
DJ
Department of Biochemistry, University of Kentucky, 800 Rose Street, Lexington, Kentucky 40536, USA. dnoonan@pop.uky.edu
Lou
Dingyuan
D
Griffith
Nicole
N
Vanaman
Thomas C
TC
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015536:Down-Regulation; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015723:Gene Library; D006801:Humans; D018192:Lymphangioleiomyomatosis / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011987:Receptors, Steroid / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014158:Transcription, Genetic; D014402:Tuberous Sclerosis / Q000235:genetics*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins
D002147:Calmodulin; D010455:Peptides; D011987:Receptors, Steroid; D012097:Repressor Proteins; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D002118:Calcium