The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains.
Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.
Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
Sangyeul
S
Han
Sun
S
Kim
Samira
S
Bahl
Lin
L
Li
Clara F
CF
Burande
Nicole
N
Smith
Marianne
M
James
Roberta L
RL
Beauchamp
Pradeep
P
Bhide
Aaron
A
DiAntonio
Vijaya
V
Ramesh
D000818:Animals; D001369:Axons / Q000378:metabolism*; D017173:Caenorhabditis elegans; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003337:Corpus Callosum / Q000378:metabolism; D004330:Drosophila; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D051379:Mice; D008817:Mice, Mutant Strains; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013569:Synapses / Q000235:genetics / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
D002352:Carrier Proteins; D044783:F-Box Proteins; C542381:Fbxo45 protein, mouse; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D011506:Proteins; C000624653:TSC2 protein, human; C000624654:Tsc2 protein, mouse; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C532954:Mycbp2 protein, mouse; D044767:Ubiquitin-Protein Ligases; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases