Structural Biochemistry/Enzyme Regulation/Amino Acid Sensing and Transporting
Amino Acids sensing
The availability of amino acids play a key role in cellular physiology my controlling signalling pathways and gene expression.
GCN2 (general control of Amino Acids biosynthesis, nondepressing 2) is a cellular sensor for amino acids sufficiency to transform protein synthesis; GCN2 can detect amino acids deficiency of uncharged aminoacyl-tRNA that is accumulated. Normally, aminoacyl-tRNA is charged.
• GCN2, a kinase capable of phosphorylating the eukaryotic translation initiation factor 2-alpha(eIF2a), which inhibits its guanine nucleotide exchange factor, eIF2B; and regulates mTORC1 and GADD34, a protein phosphatase-1 binds to TSC1 and TSC2 and promotes dephosphorylation of TSC2 T1462, a major Aktphosphorylation site, which induces TSC-GAP activity to decrease Rheb-GTP charging so that mTORC1 is inhabited.
• eIF2a indirectly impairs eIF2 function and inhibits translation initiation.(e.g.: at S51) This is upregulating the translation of a subset of mRNA. For example, ATF4 promotes expression of an array of genes for amino acids biosynthesis.
• Only leucine has forceful effects on mTORC1. Uncharged tRNA doesn’t affect mTORC1 signaling- using cell lines harboring temperature-sensitive aminoacyl tRNA synthetase activity. However, decreasing in absence of aminoacyl-tRNA doesn’t account for rapid inhibition of mRORC1 signal; so amino acids sensing by GCN2 is directly involved in AA-induced mTORC1 activation needs more evidence.
AA transporters as modulators of Intracellular AA levels
• AA transporters regulatesTORC1 by changing intracellular AA concentrations or acting as receptor/transceptor to initiate intracellular signaling pathways.
• Leucine is strong activator of mTORC1.
• System L is called LAA transporter system, which is LAT 1/2(L-type AA transporter ½ and the 4F2hc/CD98 glycoprotin) is a primary route/entrance for neutral amino acids such as leucine with flowing out of cytoplasmic amino acids. Yet, LAT1 is highly present in actively growing tumors.
• The accumulation of intracellular amino acid is done by System A transporter, such as SNAT2(sodium-coupled neutral AA transporter 2).
• SNAT2 is one of types of System A transporter, which concentrates amino acids due to its unidirectional Na+-AA-coupled transport cycle.
• The expression of both System L and System A positively correlate with mTORC1 activity. That explains glutamine stimulates leucine on TOR activity though no effect on TOR activity by itself in oocytes.
• As the result of amino acids transport, Na+ and polar/charged amino acids tend to change its polarization of cell membrane. The result of depolarization effects a rise in intracellular Ca2+, an effect implicated in AA-dependent activation of mTORC1 and Vps34.
• Insulin promotes the localization of System A AA transporter to cell surface from endosomal compartment in skeletal muscle cells in order to simulating amino acids signaling.
• Cytokines enhance System L. For example, : cytokines promote growth in lymphocytes.
• Leucine transport can possibly prevent unwanted activation of signal pathway such as TORC1 due to T-cell activation by PMA (phorbol myristate acetate) and ionomycin.
• A two-step AA transport mechanism is also proposed for mTORC1 activation by AA. First, high-affinity L-glutamine transporter, SLC1A5 is responsible for the intracellular glutamine accumulation; secondly, the heterodimeric SLC7A5/SLC3A3 bidirectional transporter that is an intracellular leucine accumulation and mTORC1 activation uses intracellular glutamine as a flowing substrate to uptake extracellular leucine.
• AA transporters in TORC1 examples: Drosophila slimfast and minidiscs sense nutrient availability and control body size. AA transporters as sensors: transceptors
• Found in lower eukaryotes such as yeast/ Drosophila.
• Yeast AA permease Gap1 activates the cAMP/PKA (protein kinase A) signaling pathway to regulate metabolism and expression of stress-responsive genes.
• Shorten Gap1 C terminus can cause permanent activation of the PKA signaling pathway independent of Gap1 transport activity (in phenotypes, constitutively active PKA in vivo).
• Ssy1 (yeast AA permease homolog) senses extracellular AA to stimulate a proteolysis-dependent signaling pathway to increase various AA and peptide permeases expression.
• Drosophila-Evidence of AA transceptor and TORC1 connection can be verified by PATH, a PAT (proton-assisted AA transporter) transporter, which regulates growth by interacting with TOR. (SLC36 is the mediator). PATH could control growth via mechanism, no need of AA transport, so it mostly acts as a receptor.
• PAT found on the surface of mammalian lysosomes, plasma membrane and endosomal compartments. It relates to AVT (AA vacuolar transporter) in yeast, so it can transport AA out of vacuole and a lysosomal equivalent structure in yeast.
• PAT express throughout the body, such as in human PAT- PAT1 and PAT4 are expressed in normal tissues and cancer cell lines strongly, are required mTORC1 activation and cell proliferation.
• The mutants in minidiscs (fat body-specific AA transporter) have normal size that promotes growth in a wild-type host.
• TORC1- a central cell-growth regulator, has to integrate a range of growth-stimulating and inhibitory signals to control translation, autophagy, and cell growth.
• PI3K-PKB/Akt-signaling branch phosphorylate TSC2 and PRAS40 in mTORC1.
• AMPK phosphorylates TSC2 and Raptor in response to energy starvation stress in mTORC1
• AA signaling is parallel to TSC1/2-Rheb branch of TORC1 regulation.
• Among all connectors between AA and TORC1, Rag GTPases are the most convincing one, which directly participate in TORC1 activation in response to AA.
• All the data supported by genetic data in yeast and Drosophila and in vitro biochemical/cell bio.
• In contrast, Vps34, MAP4K3, RalA and Rab5 signaling in AA need more verification.
• The active RagA or RagB binds Raptor to recruit TORC1 to lysosomes where TORC1 is activated by Rheb (localized in lysosomes). This explains Rheb and Rag are needed for full TORC1 activation. Rag GTPases (heterodimer) localize on the lysosomal surface to react with p14/MP1/P18 complex.
Christie GR, Hyde R, Hundal HS. Regulation of amino acid transporters by amino acid availability. Curr Opin Clin Nutr Metab Care. 2001 Sep;4(5):425–431.
- Amino Acid Signaling in TOR Activation, Joungmok Kim and Kun-Liang Guan; Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, California. Annu. Rev. Biochem. 2011. 80:1001-32. http://www.annualreviews.org/doi/abs/10.1146/annurev-biochem-062209-094414