7. SOS
• Son of sevenless
– SOS was discovered in Drosophila
melanogaster. Essential for normal eye
development in Drosophila. The SOS1 gene
encodes the Son of Sevenless 1 protein.
This protein is composed of several
important domains.
– Human：SOS1, SOS2
– GEF (guanine nucleotide exchange factors)
PxxP motif
Grb2 binding
9. Small GTPases
• A family of hydrolase enzymes, cytosolic monomers, 21 to 30
kDa.
• Homologous to the alpha subunit of heterotrimeric G-proteins,
but unlike the alpha subunit of G proteins, a small GTPase can
function independently as a hydrolase enzyme to bind to and
hydrolyze GTP to form GDP.
– GTP binding form: active
– GDP binding form: inactive
• Function as molecular switches that control many eukaryotic
cell functions including cell proliferation, cellular trafficking,
and dynamics.
• All small GTPases belong to a superfamily, often named the
Ras superfamily because the founding members are encoded
by human Ras genes initially discovered as cellular homologs
of the viral ras oncogene.
10. Regulatory Proteins of Small GTPases
• GEF (guanine nucleotide exchange factors)
– Activating GEF (Guanosine nucleotide dissociation stimulators,
GDS)
– Inhibitory GEF (Guanosine nucleotide dissociation inhibitors, GDI）
• GAP (GTPase activating protein)
Active
Inactive
11. Ras Superfamily
Subfamily Function
Ras Cell proliferation (MAPK signaling)
Rho Cellular dynamics, cell morphology
Rab Vesicular trafficking
Rap Vesicular trafficking
Arf Vesicular trafficking
Ran Nuclear transport
Rheb mTOR signaling
Rad
Rit
Miro Mitochondrial transport
>100 members，divided into 10 subfamilies based on the
amino acid sequence, structure and function.
12. Raf
• Activated by Ras
• A-Raf, B-Raf and Raf-1(C-
Raf)
• downstream effector: Ser/Thr
kinase: phosphorylates
MEK1/2
• MEK1/2 phosphorylates
ERK1/2 , which belongs to
mitogen-activated protein
kinase (MAPK) family
Comparison of human ARAF, BRAF, and
RAF1 and the worm homolog lin-45. The three
conserved regions CR1, CR2, and CR3, the
structural domains they contain (RAS-binding
domain: RBD; cysteine-rich domain: CRD; and
kinase domain), and phosphorylated residues
(positive, yellow outline; negative, red outline)
https://doi.org/10.1016/j.cell.2015.04.045
14. https://doi.org/10.1016/j.cell.2015.04.045
Raf
Functions of RAF paralogs in development and tumorigenesis
The most frequently observed mutation, a valine600 / glutamic acid substitution
(BRAFV600E), disrupts the inactive conformation of the kinase and abolishes the
requirement for dimerization, inducing persistent activation of BRAFV600E monomers
and, consequently, of the MEK/ERK pathway.
15. MAPKK/MEK Family
• 5 genes：MEK1-2, MKK3-7
• Dual-specificity kinases:
phosphorylates Ser/Thr residues.
• MEK1：
– 393aa, 44kDa
– The activity is dependent on the
phosphorylation of S218 and S222.
17. ERK1/2
• ERK2/MAPK1, mice lacking
MAPK1 have major defects in
early development.
• ERK1/MAPK3, mice lacking
MAPK3 are viable.
• ERK1/2:
– 85% sequence identity
– 42/44kDa
– The MAPK kinase of
ERK1/2 was named
MAPK/ERK kinase (MEK).
– The activation is dependent
on the dual phosphorylation
of T182 and Y184 by MEK.
18. ERK Signaling is Compartmentalized
Within the Cell
Three scaffolding proteins helps the ERK cascade transduce signals with
both high efficiency and specificity in spatial regulation.
Kinase Suppressor
of Ras (KSR)
MEK Partner 1
(MP1)
Similar Expression
to FGF Genes (Sef)
19. Duration and extent of ERK activation is controlled by
the balanced activities of MEKs and phosphatases
Dual Specificity Phosphatases (DUSPs)
Phosphatases PP2A
35. Engelman et al. Nature Reviews Genetics 7, 606–619 (August 2006) | doi:10.1038/nrg1879
Class I PI3K Signaling
• Hydrolysis of PIP3：
– 3-phosphate：PTEN
– 5-phosphate：SHIP1/2
Insulin, EGF, PDGF…
Chemokines
Grb2, IRS
Akt
A
k
t
36. Schematic Structure of PTEN
• Phosphatase and tensin homolog deleted on chromosome ten (PTEN)
• PIP2 binding motif ：6-15
• Phosphatase domain (15-185)：
– 2 Cys residues, oxidized by ROS (reactive oxygen species)
• C2 domain (186-351)：binding membrane lipid
• C-tail (352-403)
– PEST motifs (350-375): rich in Pro, Glu, Asp, Ser and Thr,
– PDZ domain-binding motif (PDZ BD)
37. PTEN is a tumor suppressor.
- Loss of PTEN function is linked to Cowden syndrome (
多发性错构瘤综合征) in which patients have a
predisposition to malignancies such as prostate cancer,
glioblastoma, endometrial tumors, and small-cell lung
carcinoma.
- PTEN-negative LNCaP cells
38. Phosphorylation of Akt
• Also called PKB
• Kinases that phosphorylate Akt：PDPK1/PDK1，mTORC2/
PDK2
• Akt1: T308 /S473, phoshorylation of T308 is critical for Akt
activation, 480aa
Akt1
Akt2
Akt3
Ser/Thr Kinase RD
480aa
481aa
479aa
40. PDPK1/PDK1
• PDPK1 (3-phosphoinositide dependent protein kinase-1) or
PDK1, phosphorylates Thr308 of Akt
– N-terminal Ser/Thr kinase domain
• the substrate binding site
• the ATP binding site
• the docking site
– C-terminal PH domain: the affinity for PI(3,4,5)P3 and
PI(3,4)P2 is much higher than the PH domain of Akt.
41. mTORC2/PDK2
• Mammalian target of rapamycin complex 2
• Complex：
– mTOR
– LST8/GβL(G-protein β-subunit like protein)
– Rictor (rapamycin-insensitive companion of mTOR)
– mSIN1(mammalian stress-activated protein kinase
interacting protein 1)
• Ser/Thr kinase，also known as PDK2:
– Phosphorylates Ser473 of Akt
42. Dephosphorylation of Akt
• pThr308: PP2A 蛋白磷酸酶2
• pSer473: PHLPP(PH domain
and Leucine rich repeat Protein
Phosphatases), PP2C
– PHLPP1: Akt2, Akt3
– PHLPP2: Akt1, Akt3
44. IRS Mediates IR Induced PI3K Signaling
Berridge MJ. Cell Signalling Biology. 2014. doi:10.1042/csb0001002
IRS: insulin receptor substrate
45. Binding of insulin to its receptors leads to the activation of AKT2.
One of its substrates is glycogen synthase kinase-3β (糖原合酶激酶-3β), which
becomes inactivated, unable to phosphorylate and inactivate glycogen synthase.
Also, the phosphatase ensures rapid dephosphorylation and
activation of glycogen synthase allowing glycogen synthesis to recommence.
In addition, phosphorylase (磷酸化酶) is inactivated by dephosphorylation.
Glycogenolysis is arrested.
47. Conservation of PI3K Signaling
• PI3K signaling is highly conserved in evolution from the
nematode C. elegans to humans.
IR
PI3K
PTEN
FOXO
FOXO
apoptosis
C. elegans
Humans
52. Ras Superfamily
Subfamily Function
Ras Cell proliferation (MAPK signaling)
Rho Cellular dynamics, cell morphology
Rab Vesicular trafficking
Rap Vesicular trafficking
Arf Vesicular trafficking
Ran Nuclear transport
Rheb mTOR signaling
Rad
Rit
Miro Mitochondrial transport
>100 members，divided into 10 subfamilies based on the
amino acid sequence, structure and function.
53. Small GTPase Rheb
• Rheb: Ras homolog enriched in brain
• A member of the small GTPase superfamily and encodes a
lipid-anchored, cell membrane protein with five repeats of
the RAS-related GTP-binding region.
• Rheb shuttles between a GDP-bound form and a GTP-
bound form, and farnesylation of the protein is required
for this activity.
54. Regulation of Rheb Activity
• Tuberous sclerosis complex
(结节性硬化症), tumor
repressor
• Upstream of Rheb, key
regulator of mTOR signaling
– TSC1 (hamartin)
– TSC2 (tuberin)
• Stimulates the GTPases
activity of Rheb (GAP)
GEF?
55. TSC1 and TSC2
• Tuberous sclerosis (TSC) was first described by Désiré-Magloire Bourneville in
1880 (and called Bourneville’s disease in France).
• TSC is a rare (30–100 per million, worldwide), multisystem genetic disease
characterized by the growth of benign tumors (良性肿瘤) in the brain and in other
vital organs including the kidneys, heart, eyes, lungs, and skin.
• TSC is caused by mutations on either of 1 or 2.
• TSC1 and TSC2 are discovered as loss-of-function mutants in patients suffering
from tuberous sclerosis.
• TSC1/2 genes encode for the proteins hamartin and tuberin, and act as tumor
suppressors, regulating cell proliferation and differentiation.
59. p mTORC1 integrates information about nutritional abundance and environmental
status to tune the balance of anabolism and catabolism in the cell.
p mTORC2 governs cytoskeletal behaviour and activates several pro-survival
pathways.
p mTORC1 can be inhibited by rapamycin.
p mTORC2 responds only to chronic rapamycin treatment.
mTORC1 and mTORC2 regulation
https://doi.org/10.1038/s41580-019-0199-y
60.
61. mTORC1 can activate several proteins by phosphorylation, such as the p70 S6K
kinases and the EIF4E-binding protein 1 (4EBP1).
Downstream signaling of mTOR
62. Substrates of mTORC1
• p70-S6 Kinase 1 (S6K1)
– Ribosomal protein S6
kinase 1, MW 70kDa
– Ser/Thr kinase
– mTORC1 phosphorylates
T389 of S6K1 (key step)，
then PDK1 phosphorylates
T229, completely
activating S6K1
– Activated S6K1
phosphorylates S6 (S235,
S236, S240, S244),
resulting in enhanced
protein synthesis and cell
proliferation.
Murphy E , Steenbergen C. Physiol Rev 2008;88:581-609
63. Substrates of mTORC1
• 4E-BP1(the eukaryotic initiation
factor 4E binding protein 1，真核
起始因子4E结合蛋白1)
– Nonphosphorylatled 4E-BP1
associates tightly with eIF4E,
therefore inhibits the
association of eIF4E with
mRNA and translation.
– mTORC1 phosphorylates 4E-
BP1 at multiple sites (T37,
T46, S65, T70), p4E-BP1
disassociates with eIF4E, and
free eIF4E associates with
mRNA, promoting the
formation of translation
initiation complex.
69. p The intracellular localization of mTOR complexes, particularly of mTORC1 under amino acid starvation, has not been
clearly defined.
p Although PI3K acts upstream, our understanding of the molecular mechanism of mTORC2 activation is not satisfactory.
p Our knowledge of amino acid sensing to activate mTORC1 is far from complete. More work is needed to determine
whether many of the proposed mechanisms are physiologically relevant.
p As a central cell growth controller, mTORC1 has to integrate a wide range of intracellular and extracellular signals, but
signal integration and crosstalk are not fully understood.
Questions about mTORC1
Help to advance our understanding of basic biology, as well as provide scientific bases for treatments against various
human pathologies, such as metabolic disorders, cancers, neurodegeneration, immune diseases and ageing.
70. One of the greatest challenges presently facing the field is resolving the intracellular localization of TOR
complexes. It has long been suspected that there are functionally distinct pools of mTOR complexes and that
intracellular localization may compartmentalize their activity. Advances in live-cell imaging and
visualization of tagged proteins will provide the necessary tools to address this challenge.
