Pam (Protein associated with Myc) functions as an E3 ubiquitin ligase and regulates TSC/mTOR signaling.
The tumor suppressor tuberin, encoded by the Tuberous Sclerosis Complex (TSC) gene TSC2, negatively regulates the mammalian target of rapamycin (mTOR) pathway, which plays a key role in the control of cell growth and proliferation. In addition to naturally occurring mutations, several kinases including Akt, RSK1, and ERK are known to phosphorylate and inactivate tuberin. We demonstrate a novel mechanism of tuberin inactivation through ubiquitination by Pam, a putative RING finger-containing E3 ubiquitin (Ub) ligase in mammalian cells. We show that Pam associates with E2 ubiquitin-conjugating enzymes, and tuberin can be ubiquitinated by Pam through its RING finger domain. Tuberin ubiquitination is independent of its phosphorylation by Akt, RSK1, and ERK kinases. Pam is also self-ubiquitinated through its RING finger domain. Moreover, the TSC1 protein hamartin, which forms a heterodimer with tuberin, protects tuberin from ubiquitination by Pam. However, TSC1 fails to protect a disease-associated missense mutant of TSC2 from ubiquitination by Pam. Furthermore, Pam knockdown by RNA interference (RNAi) in rat primary neurons elevates the level of tuberin, and subsequently inhibits the mTOR pathway. Our results provide novel evidence that Pam can function as an E3 Ub ligase toward tuberin and regulate mTOR signaling, suggesting that Pam can in turn regulate cell growth and proliferation as well as neuronal function through the TSC/mTOR pathway in mammalian cells.
Han
Sangyeul
S
Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
Witt
Rochelle M
RM
Santos
Túlio M
TM
Polizzano
Carolyn
C
Sabatini
Bernardo L
BL
Ramesh
Vijaya
V
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases; D014402:Tuberous Sclerosis / Q000235:genetics; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
D048868:Adaptor Proteins, Signal Transducing; C000624653:TSC2 protein, human; C000624655:Tsc2 protein, rat; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D006899:Mixed Function Oxygenases; D044763:Ubiquitin-Conjugating Enzymes; C114022:MYCBP2 protein, human; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases; C546842:MTOR protein, human; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases