Ch. 15 The Insulin Signaling
Network and Insulin Action
2004. 6. 22
Insulin signaling at target tissue
 Normal growth and development
 Normal homeostasis of glucose, fat, and protein metabolism
Improved understanding of signaling pathways involving insulin
action
 Better understanding of pathophysiology of insulin resistance
asscociated with obesity and type 2 diabetes
 Newer and more effective therapeutic agents
This chapter


Current understanding of insuin action
 Strucutre of IR, elements that constitute insuling signaling
pathways
Impairments in signaling pathways and new paradigms regarding
the molecular basis of insulin resistance
Insulin receptor and its substrates
STRUCTURE OF
INSULIN RECEPTOR
Family of homologous receptor tyrosine kinases (RTKs)
- IR / IGF-1R(insulin-like growth factor-1)
- IRR (insulin receptor related receptor) : orphan receptor
Proteolytic cleavage of a single polypeptide chain precursor
→ α & β chains linked by disulfide bonds
→ biologically active α2β2 receptor heterotetramer
Product of a single gene containing 22 exons
- 1,355 amino acids, M.Wt of 153,917
- Exon 1 – 11 : α subunit / Exon 12-22 : β subunit
- Alternative splicing of exon 11 in hematopoietic tissues
- Longer transcript predominates in liver, muscle, and adipose tissues
Two IR isoforms
IR type A (IR-A or Ex11-)



(-) Sequence coding for 12 a.a. in C-terminus of α chain of Rc.
Preferentiallly activates type Ia of PI3-kinase and p70S6K
Promotes insulin gene transcription in pancreatic β cells
IR type B (IR-B or Ex11+)



(+) Sequence coding for 12 a.a. in C-terminus of α chain of Rc.
Stimulates class II of PI3-kinase and PKB (Akt)
Promotes glucose/insulin stimulated β-glucokinase transcription
Different localization of two IR isoforms in plasma memb.
of β cells and different sensitivity for insulin
The Extracellular Domain
Entire α-subunit and about one third of β-subunit
Ligand-binding domains, with α-subunit being the
primary lignand-binding site
Analogous region of IR (residues 1-468)
Cannot bind ligand on its own, high-affinity insulin binding
 Restored by addition of residues 704 to 719 of IR
⇒ carboxyl-terminal region of α-subunit
: part of ligand- binding site

Three repeats of fibronectin type III domain (FnIII)
Deletion of residues 450 to 601
: blunted Tyr autophosphorylation response
⇒ involved in transmission of insulin-binding signal to tyrosine
kinase

N-linked and O-linked sugar moieties
Mutations of a.a. of potential sugar binding sites
: alter IR kinase acitivity
 Improper glycosylation : accelerates IR degradation
⇒ required to preserve proper structure and function of IR

The Transmembrane Domain
Part of β-subunits
α-helix structure with seven turns
Function primarily as passive lipid anchor
23 hydrophobic a.a., flanked at C-terminal end by a short
sequence of basic residues (Arg-Lys-Arg)


Potential interactions with head group of negatively
charged phospholipids
May represent a stop-transfer signal, anchoring IR in
membrane during biosynthesis
The Intracellular Region
C-termianal 403 a.a. of β-subunits,
3 subdomains
1. Juxtamembrane (JM) domain
At least one autophosphorylation site (Tyr 972) in NPXY motif
- Binding site for IR substrates such as Shc and IRS proteins
Regulation of IR internalization (NPXY, GPLY, di-Leu motifs)
Replacement of Tyr972 with Phe or Ala
- Inability of mutant Rc to interact with substrate proteins (IRS-I & Crk-II)
→ Impairs receptor signal transmission and both metabolic and growthpromoting effects of insulin
- Rescued by overexpression of IRS-1
⇒ Involved in interactions of Rc with substrate proteins
2. Tyrosine kinase domain of IR : hallmark of RTK family


84% homology with IGF-1R
Two lobes with a single connection

N-terminal lobe : ATP-binding site

C-terminal lobe
: active site (catalytic loop), three autophosphorylation
sites (activation loop), and kinase-insert region
3. C-terminus domain of IR



Two autophosphorylation sites, still unresolved role
Mutations of these Tyr residues

Augments insulin-dependent activation of MAPK and PI3-K

Impaired metabolic effects and impaired induction of c-fos but
augmented mitogenic signaling
Negatively regulate growth-promoting effects of insulin
INSULIN ACTION AT THE
CELLULAR LEVEL
Diverse biologic responses by binding of insulin to IR




Glucose transport
Glycogen and protien synthesis
Mitogenensis
Cell survival
Ligand Binding and Receptor
Autophosphorylation
Binding of insulin to specific regions of α-subunit
→ rapid conformational change in Rc
→ activation of tyrosine kinase domain
(transautophosphorylation)
: kinase domain in one half of the receptor-dimer
phosphorylates cytoplasmic tyrosine residues in
activation loop of the other half of the receptor-dimer
→ activation loop swings out of the catalytic site to give
unrestricted access to ATP and substrates
Insulin Receptor Substrates
Insulin receptor substrates


Three isoforms of Shc, IRS proteins (IRS-1 to -4), p60dok, Cbl,
APS, and Gab-1
Tyr-phosphorylated IR substrates by activated IRK
: Function as signaling scaffolds, providing a docking
interface for proteins having SH2 domains
SH2 proteins


P-Tyr phosphatase SHP2 (SH-PTP2) and cytoplasmic Tyr
kinase Fyn : Enzymes
p85 regulatory subunit of PI3-kinase, Grb2, or APS

Function as adaptor proteins for downstream effectors that further
propagate the metabolic and growth promoting effects of insulin
IRS proteins
Conserved pleckstrin homology (PH) domain
1) Membrane - lipid interactions


Anchor IRS proteins to membrane phosphoinositides
Helps to localize IRS proteins in close proximity to Rc
- deletion of PH domain : ↓ tyrosine phosphorylation of IRS-1
2) Protein – protein interactions
PH domain – interacting protien (PHIP) selectively binds

Overexpression of PHIP
: ↑ insulin-induced transcriptional responses

Dominant-negative mutant of PHIP (DN-PHIP)
: specifically blocks transcriptional & mitogenic signals by insulin
: inhibits actin cytoskeletal reorganization and translocation to
membrane of GLUT-4
⇒ PHIP : physiologic binding protein of IRS-1 PH domain, which
plays a role in insulin signaling

P-Tyr binding (PTB) domain of IRS proteins


75% sequence identitiy between IRS-1 and IRS-2
Function as a binding site to NPXY motif of JM region of IR
C-terminal region of IRS proteins

Contains multiple Tyr-phosphorylation motifs that serve as
docking sites for SH2 domain-containing proteins (p85α
regulatory subunit of PI3-kinase, Grb2, Nck, Crk, Fyn, SHP-2…)
> 70 potential Ser/Thr phosphorylation sites

Several kinases that can phosphorylate IRS-1
: casein kinase II, glycogen synthase kinase 3, MAPK, PI 3-kinase

IRS-1



Insulin (-) : Ser (strong) and Tyr (weak) phosphorylation
Insulin (+) : ↑ Tyr and Ser phosphorylation
Phosphorylation of IRS-1 on Ser/Thr residues


reduces its ability to undergo Tyr phosphorylation by IRK
serves to shut off insulin signaling
Relative role of IRS proteins in mediating insulin action
1) IRS-1 and IRS-2
 IRS-1 knockout mice : generalized growth retardation, as well
as insulin resistance and IGT, compensation of IRS-2 (PI3-K)
 IRS-2 null mice : insulin resistance but growth defects limited
only to pancreatic β-cells → type 2 diabetes
 Complementary rather than redundant role in insulin signaling
 Attributed to selected structural differences, in addition to
differences in their tissue distribution & subcellular localization
2) IRS-3 and IRS-4
 Act as negative regulators of IRS-1 and IRS-2 in cultured cells
 IRS-3 and IRS-4 knoukout mice : mild defects in growth and
metabolism
Shc family
Adapter proteins that serve as major substrates of IRK
Three isoforms of 46, 52, and 66 kd
Insulin-stimulated Tyr-phosphorylated Shc + SH2 domain of Grb2
→ activation of mSOS/Ras/MAPK signaling pathway
PTB & SH2 domain : Interaction with JM or C-terminal of IR
PI 3-kinase as a necessary intermediary step facilitating insulinstimulated Tyr-phosphorylation of Shc through PTB domain
PP2A (Ser/Thr phosphatase)
: inhibit Tyr-phosphorylation of Shc independent of P-Tyr binding
Gab-1 (Grb2-associated binder-1)
Insulin receptor substrate
Tyr-phosphorylated Gab-1 recruits downstream signaling
elements possessing SH2 domain (p85 PI 3-kinase,
phospholipase C-γ, SHP-2 (protein tyrosine phosphatase), and Crk)
Substrate for other RTKs (EGF, FGF, HGF, NGF Rcs)
Physiologic roles of Gab-1 : little known


May be part of signaling pathways leading to cell growth,
transformation, and apoptosis
Mediator of osmotic shock signal transduction pathway
that induces glucose transport in adipocytes through Gab1-associated PI 3-kinase acitivity
The Insulin Signaling Network
Three major pathways
: PI 3-kinase, MAPK, and
Cbl/CAP pathways
MAPK pathway
: general signaling pathway
leading to enhanced cell
growth
PI 3-kinase and Cbl/CAP
pathways
: biologic responses that are
more unique to insulin
action
Insulin signaling network
• Activated
IRK
• Substrates
• 3 signaling
pathways
DNA / RNA / Protein
Synthesis
Gluconeogenic
Gene Transcription
Glycogen
Synthesis
Glucose
Transport
• Function
The PI 3-kinase pathway
Central role of metabolic & growth-promoting actions of insulin
p110 catalytic subunit and p85 regulatory subunit
Direct interactions of regulatory subunit with Tyr-phosphorylated YMXM
and YXXM motifs of activated growth factor receptors, or with adapter
proteins such as IRS proteins → activation of PI3K
 p110 catalytic subunit in close proximity to its lipid substrates in cell
membrane
 May relieve an inhibitory effect of p85 on p110 kinase activity
p85α : predominant subunit that mediates most biologic responses to
insulin (at least eight isoforms of regulatory subunits)
Inhibitors of class Ia PI 3-kinase, or transfections with dominant
negative constructs of enzyme block most metabolic actions of
insulin including stimulation of glucose transport, glycogen, and
lipid synthesis.
Monomeric p85 inhibits IRS protein-mediated signal by competing
with p85-p110 dimer.
 Partial depletion of p85 improves insulin signaling
 Complete depletion of p85 results in a significant decrease in PI 3kinase mediated biologic responses
⇒ optimal signaling depends on a critical molecular balance between
regulatory and catalytic subunits
Association of p85-p110 complex with IRS molecules
→ production of PIP3
→ interaction with PH domain of PDK1, PKB, and other signaling
molecules
→ their recruitment to plasma membrane
→ changes in their structure, function, and their substrate availability
Ser kinase activity independent of generation of PIP3
Atypical Protein Kinase C Isoforms :
PKCζ and PKCλ
Activated by PI 3-kinase and PDK1
Increase basal and insulin-stimulated translocation to
the membrane of GLUT-4 in adipocytes and muscle cells
Play a critical role in physiologic negative feedback
control mechanism, induced by insulin, that serves to
terminate insulin action
: PKCζ-mediated phosphorylation of IRS proteins leads
their dissociation from IR, thereby terminating insulin
signaling
Protein Kinase B (Akt)
Major substrates of PDK1
Mediating regulation of glucose transport, glycogen
synthesis, protein synthesis, antilipolytic effects of insulin, as
well as cell growth and cell survival induced by insulin
Phosphorylation at Thr308 by PDK1 & at Ser473 by PDK2
→ maximal activation of PKB
Three PKB isoforms

PKB1/PKBα : required for normal growth

Deletion of Aktβ(PKB2) : hepatic insulin resistance

Defect in a ability of insulin to activate PKB2 and -3 but not PKB1
: impaired insulin-stimulated glucose transport
Protein kinase B, Glucose Transport,
and Glycogen Metabolism
PI3K
Regulates translocation of GLUT-4
: obscure mechanism
PDK1
Important role in insulin-induced glycogen synthesis
: phosphorylation and inactivation by PKB of GSK3β
PKB
(phosphorylates and inactivates glycogen synthase
(GS))
GSK3β
→ promotes GS activity and glycogen synthesis in
response to insulin
GSK3β Inactivates by phosphorylation protein
eIF-2B
GS
synthesis eukaryotic initiation factor (eIF)-2B
→ Insulin-mediated activation of PKB reverses, thereby
enhancing protein synthesis
DNA / RNA / Protein
Synthesis
Glycogen
Synthesis
Protein kinase B and Protein Synthesis
Mammalian target of rapamycin (mTOR)
- Ser/Thr kinase that serves as a molecular sensor that regulates
protein synthesis on the basis of nutrients availability
Mechanisms of activation of insulin-induced protein synthesis
PI3K
at translational level
PDK1
P70 S6K
PKB
mTOR
ribosomal protein
S6
4E-BP1
eIF-4E
DNA/RNA/Protein Syntheis
: translation repressor
Protein kinase B, Gene Expression,
and Cell survival
Insulin inhibits nuclear translocations of transcription factors by
phosphorylation these factors by PKB in an insulin-dependent manner
Forkhead family (FH)
 FKHR (Foxo-1, forkhead box transcription factor O1), FKHRL1, AFX
 Insulin-mediated phosphorylation of Foxo-1 by PKB
→ interaction of Foxo-1 with 14-3-3 family of proteins
→ retention of Foxo-1 in cytoplasm and prevents Foxo-1 from
translocating to the nucleus
→ Inhibition of expresssion of a number of Foxo-1-regulated genes
: gain of function Foxo-1 mutation targeted to liver & pancreatic β-cells
→ increased hepatic glucose production and impaired β-cell
compensation due to decreased Pdx 1 expression → type 2 diabetes
 Foxo-1 functions in adipose cells to couple insulin signaling to
adipogenesis : switching preadipocytes from proliferation to terminal
differntiation
Antiapoptotic functions of insulin and IGF-1 by PKB


PKB phosphorylation of apoptosis-inducing protein Bad
→ creates binding sites for 14-3-3 proteins and
prevents Bad from binding to Bcl-2 family members,
Bcl-2, Bcl-XL, thus releasing them for a cell survival
response
PRAS40
: novel substrate of PKB
: phosphoylation of PKB → binding of PRAS40 to 14-3-3
The Mitogen-Activated Protein
Kinase Pathway
MAPK kinase kinase (MKKKs) → MAPK kinase (MKK)↑
→ MAPK↑
Three well-characterized subfamilies of MAPKs



Extracellular signal-regulated kinases ERK1 and ERK2
c-Jun NH2-terminal kinases JNK1, JNK2, and JNK3
four p38 enzymes p38α, β, γ, δ
ERK1 and ERK2
Activated ERKs mediate growth-promoting effects of insulin by
phosphorylating transcription factors such as Elk-1, leading to induction
of gene expression
Magnitude and duration of ERK activation
 Critical determinants of final cell type-specific physiologic outcome
 Regulated by balance of both activating kinases and inactivating
phosphatases
Insulin-induced adipocyte differentiation by ERK
 Activation of MEK/ERK pathway at late stages of adipogenesis
: likely to block adipogenic gene expression due to MAPKdependent phosphorylation of PPAR-γ
 Activation of pathway early during adipogenesis before PPAR
expression might promote differentiation by activating transcription
factors operating to initiate PPAR and C/EBP expression
- Insulin : principal regulator of MAPK pathway in preadipocytes and
promotes their differentiation by enhancing expression of C/EBPα
and PPAR-γ
C-Jun N-terminal Kinase
Stress-activated protein kinases on the basis of their
activation in response to inhibition of protein synthesis
Bind and phosphorylate c-Jun (component of AP-1,
important regulator of gene expression) and increase its
transcriptional activity
Insulin stimulation
→ binding of JNK to IRS-1 and phosphorylation on Ser307
→ inhibition of insulin signaling
Dual function as a heterologous inhibitor of insulin
action during acute and chronic inflammation and as a
negative feedback regulator of insulin action by
phosphorylating Ser307 in IRS-1
p38 Kinases
Activated by inflammatory cytokines, hormones, ligands
for GPCRs, and stresses such as osmotic or heat shock
Undergoes insulin- and IGF-1-dependent activation



PI 3-kinase-mediated activation of PKB and mTOR
PI 3-kinase-mediated activation of Rac and p21-activated
kinase (PAK)
Insulin-stimulated activation of MKK3/6
Role of p38


Promoting differentiation of cultured muscle cell lines
through phosphorylation of MAPK AP-2, degradation of
IκBα, and induciton of NFκB
Mediates insulin-induced GLUT-4 transport activity,
rather than GLUT-4 translocation
The CAP/TC10 pathway and Glucose
Transporter 4 Translocation
• Adapter proteinProtein(CAP)/TC10 pathway
Cbl/Cbl-Associated
• Expressed in insulin-sensitive tissue
P-Tyr IR
• Markedly induced during adipocyte diff.
→ APS
• Increased expression by PPARγ agonists
→ P-Tyr Cbl
→ Cbl-CAP complex
(3 SH3 domain)
→ lipid raft, flotillin
(SoHo domain)
→ P-Tyr Cbl-CrkII
→ CrkII-C3G complex
→ activation of TC10
Constitutive activation of TC10 by overexpression of C3G
: not mimic insulin action but potentiate action of insulin
Active mutant of PI 3-kinase : fully insulin action
Two pathways : synergistic signals in regulation of glucose transport
TC10
Activation of TC10 : specific for insulin
Disruption of its activation : blocks insulin-stimulated glucose
transport and GLUT-4 translocation
Posttranslational modification (farnesylation and palmitoylation)
of TC10 → secretory pathway → targeting to lipid raft domain
Downstream effectors that couple TC10 to GLUT-4 translocation
 Exo70 (a component of exocyst complex)
Targeting of GLUT-4 vesicle to plasma membrane, site of fusion
 Proximity with N-ethylmaleimide sensitive factor (NSF) attachment
protein (SNAP) receptor complex involved in docking and fusion
Rab protein (Rab 4) : might mediate this tethering step
Cdc42-interacting protein 4/2 (CIP4/2) : intracellular compartment
→ plasma membrane upon insulin stimulation



TC10 seems to stimulate GLUT-4 translocation in a PI 3-kinaseindependent manner
Cdc42



GTPase, 69% homology and 86% similarity to TC10
Downstream effector of Gαq/11 and upstream regulator of
PI3-kinase and PKCλ in insulin-stimulated pathway leading to
GLUT-4 translocation
promote glucose transport by activating PI3-kinase pathway
Regulation of GLUT-4 translocation by TC10 & Cdc42

Reorganization of cytoskeleton cortical actin


Engagement of molecular motors, Myo1c


Rho family GTPases : control formation of actin stress fibers
lamellipodia and filopodia
Controls movement of intracellular GLUT-4-containing vesicles to
plasma membrane
Proper structural organization of plasma membrane caveolin
and functional clathrin : regulate the rate of endocytosis of
GLUT-4, thus affecting the overall rate of GLUT-4 recycling
- IRK directly catalyzes tyrosine phosphorylation of caveolin
INHIBITION OF INSULIN
RECEPTOR SIGNALING
Terminate insulin’s effect immediately following insulin
stimulation


Through action of lipid and protein phosphatases
Through activation of Ser/Thr kinases that phosphorylate
and uncouple various elements in insulin signaling pathways
Longer time scale

Reduction in cellular component of IR, its substrates, and
other signaling elements
Insulin Receptor Internalization
and Degradation
Rapid internalization of IR following insulin binding
→ either to their degradation or recycling to cell surface
Surface redistribution
→ progressive concentration of receptor-insulin complex in
clathrin-coated pits (internalization gates)
Stimulation of intrinsic Tyr kinase activity of IR following
insulin binding is a prerequisite for surface redistribution
IR internalization independent of its ability to phosphorylate IRS
proteins but impaired internalization of IR associated with
impaired phosphorylation of Shc
Effect of ECM proteins on receptor internalization



Interactions of different ECM proteins with cell surface integrins :
affect the rate of IR endocytosis
Specific polymerized actin structures in the form of filamentous
actin tracks : required for maintain proper IR internalization
Independent of effects of ECM proteins on IRK activity and its
ability to phosphorylate downstream effectors (IRS proteins)
Insulin signaling and insulin responsiveness are dually
regulated by the adhesive properties of the cells
- Ligation of ECM proteins by cell surface receptors (integrins)
 Generates signaling cascades that modulate the activity of IRK
 Dictate the rate and extent of IR internalization & degradation
Role of Protein & Lipid Phosphatases
Lipid Phosphatases
Phosphatidyinositol-3-phosphatases


Phosphatase and tensin homologue (PTEN)
SH2 domain-containing inositol 5-phosphatase SHIP2
Overexpression of PTEN or SHIP2
→ decreased levels of PIP3
→ might terminate signal transduction or change the
nature of phosphoinositides, altering binding specificity
Protein Tyr Phosphatases (PTPs)
Prominent role in negative regulation of IR signaling
- Dephosphorylate the IR and its substrates and thus serve to
terminate IR signaling
Transmembrane receptor-like PTPs LAR and PTPα
 LAR : dephosphorylating adapter proteins such as IRS-2
 PTPα : not alter phosphorylation status of IR, IRS-1, or Shc
Endoplasmic reticulum-associated PTP1B and its close
homologue TCPTP




Function as direct IR phosphatases, May act in concert
PTP1B in complex with IRβ subunit activation loop, encompassing
pTyr residues at 1162 and 1163
Insulin stimulates Tyr phosphorylation & inactivation of PTP1B
PTP1B may act as a point of counterregulation of insulin action by
catecholamines (↑cAMP & activation of PKA & PTP1B)
Ser Phosphorylation as a Regulatory
Means to Terminate Insulin Signaling
Control mechanisms

Homologous desensitization (Autoregulation)
- Downstream enzyme inhibit upstream elements

Heterologous desensitization
- Signals from apparently unregulated receptor pathways
can inhibit the signal
Ser/Thr Phosphorylation of the
Insulin Receptor
IR



Basal state : both P-Ser and P-Thr but no P-Tyr residues
Insulin (+) : abrupt increase in P-Tyr content of Rc
→ slower increase in its P-Ser and P-Thr content
IRs with P-Ser residues in basal state
: Tyr autophosphorylation more slowly, or even not at all
Ser/Thr phosphorylation
: act as a physiologic feedback control mechanism to inhibit
insulin-stimulated Tyr phosphorylation of Rc
Phosphorylation of Ser955/956, Ser1293/1294, Thr1336
 Stimulated by insulin, but not major regulatoy sites
Ser/Thr kinases : largely unknown, phosphorylation of IR on Ser1078
without affecting its Tyr kinase activity
Phosphorylation of the Insulin Receptor
Induced by Protein Kinases A and C
Treatment of cells with inducers of PKA



Ser/Thr phosphorylation of IR
Impairs the ability of IR to function as a Tyr kinase
Contribute to catecholamine-mediated insulin resistance
Several PKC isoforms




Mediate Ser/Thr phosphorylation of IR in intact cells
Inhibit receptor autophosphorylation
Not inhibit the receptor tyrosine kinase
Further classification for physiologic role of a direct
phosphorylation of IR by PKC
Ser/Thr Phosphorylation of the Insulin
Receptor and Insulin Resistance
Insulin resistance associated with Type 2 diabetes, obesity,
hypertension, chronic infection, and C-V dis.
Potential mechanism for induction of insulin resistance
: Excessive Ser phosphorylation of IR

Polycystic ovary syndrome (PCOS)


Decrease in IR autophosphorylation
Significant increase in Ser phosphorylation of Rc β subunits and
decreased insulin-induced Tyr phosphorylation of IR (50%)
Increased Ser phosphorylation of IR


decreases its Tyr kinase activity
One mechanism for defect in insulin action in PCOS and
other forms of insulin resistance
Ser/Thr Phosphorylation of Insulin
Receptor Substrates Proteins
Key negative feedback control mechanism
 uncouples IRS proteins from their upstream and
downsteam effectors
 terminates signal transduction in response to insulin
TNF-α, free fatty acids and cellular stress
→ Activation of Ser/Thr kinases
→ Ser/Thr phosphorylation of IRS protein
→ Inhibition of insulin signaling and
induction of insulin resistance
Insulin-Stimulated Ser/Thr Phosphoryation
of Insulin Receptor Substrate Proteins : Dual
PKCζ
PKB : Protects IRS proteins from rapid action of PTPs
enables IRS proteins to maintain Tyr-phosphorylated acitive conformation
Mechanisms of inhibition of insulin-stimulated Tyr phosphorylation
of IRS proteins by Ser/Thr phosphorylation of IRS proteins
(a) Releases IRS proteins from
intracellular insoluble
multiprotein complexes
including cytoskeletal elements
(b) Dissociation of IRS proteins
from JM domain of IR
(c) Inhibits the ability of
downstream effectors such as
PI3K to dock and bind to
specific Tyr residues at Cterminal tail of IRS proteins
(d) Turns IRS proteins into
inhibitors of IRK
(e) Induces degradation of IRS
proteins
Downstream effectors of PI 3-kinase as negative regulator of IRS
protein function
 mTOR :↑ phosphorylation of Ser residue at C-terminus of IRS
→ inhibits insulin-stimulated Tyr phosphorylation of IRS-1 and its ability to
bind PI 3-kinase

PKCζ : phosphorylation of IRS proteins
→ dissociates IR-IRS complexes and inhibits the ability of IRS proteins to
undergo insulin-stimulated Tyr phosphorylation
: function as an insulin-stimulated IRS kinase

IKKβ : Ser/Thr kinase (degradation of IκB → activation of NFκB)
Serves as substrate for PKCζ and activated by PKCζ
 Insulin-stimulated IRS kinase
- ↓ insulin-stimulated Ser phosphorylation of IRS-1 by salicylates
- IKKβ stimulated by stress inducers

: phosphorylation of IRS-1 on Ser307: adjacent to PTB domain, disruption
of interaction of between JM domain of IR and PTB domain of IRS-1
JNK : Direct interaction with IRS-1 and phosphorylation of IRS-1 at Ser307
in an insulin-dependent manner (similar to IKKβ)
Ser/Thr Phosphoryation of Insulin Receptor
Substrate Proteins and Insulin Resisitance
Inducers of insulin resistance (phorbol esters, FFA, TNF-α)
: ↑ Ser/Thr phosphoryation and ↓insulin-stimulated Tyr
phosphorylation of IRS Protein (pathologic condition)
TNF-α : activation of PKCζ & its downstream target IKKβ
Induction of sphingomyelinase and production of ceramide
→ stimulation of PKCζ activity
 Induction of complex formation btw PKCζ, p62, and RIP proteins
→ link PKCζ to TNF-α signaling
(adaptors of TNF-α Rc)

IKKβ : IRS kinase



Stimulated by FFAs, proinflammatory cytokines, and other inducers of
insulin resistance
Activation or overexpression of IKKβ : attenuates insulin signaling
Inhibition of IKKβ by high doses of salicylates : ↑glucose metabolism


Mechanism of activation of IKKβ by FFAs
: FFA-derived metabolites (diacylglycerol and ceramide)
→ activation of PKCθ and PKCζ → activation of IKKβ
Inhibition of IKKβ prevents Ser/Thr phosphorylation of IRS proteins by
high-fat diet, TNF-α, or phosphatase inhibitors
⇒ IKKβ as a point of convergence
Ser kinases downstream of insulin signaling and
Ser kinases activated by proinflammatory cytokines such as TNF-α
activate IKKβ to inhibit insulin signaling both under physiologic and
pathologic conditions
JNK


activated by proinflammatory cytokines, abnormally elevated in obesity
IRS kinase that phosphorylate Ser307, to uncouple IRS-1 from IR
PKCθ : mediator of FFA-induced insulin resistance

phosphorylation of IRS-1 at Ser307
PKCα : ↑ by phorbol esters or endothelin-1, phosphorylation at Ser612
GSK3, casein kinase II, novel kinase that phosphorylates IRS at Ser789
Phosphorylation-Mediated Degradation
of Insulin Receptor Substrate Protein
Ser phosphorylation of IRS proteins
: mainly short-tem mechanism to inhibit insulin signaling
Ubiquitin/proteasome-mediated degradation of IRS proteins
: long term insulin resistance
Ser/Thr phosphorylation via PI3-kinase/Akt/mTOR signaling pathway
 Phosphorylation of Ser307, in close proximity to PTB domain
(uncoupling interaction of IRS-1 with IR/ targeting IRS-1 to degradation)
 N-terminal region of IRS-1, including PH and PTB domain
 Suppressors of cytokine signaling (SOCS) proteins
 ↑ by proinflammatory cytokines & inducers of insulin resistance
 SOCS1/3 bind to elongin BC-containing E3 ubiquitin-ligase
complex via conserved C-terminal SOCS box
→ promote ubiquitination and
degradation of IRS1 & IRS2

Ser/Thr Phosphorylation of Shc Proteins
Chronic stimulation with insulin
→ persisitent phosphorylation of mSOS by MAPK
→ dissociation mSOS from adaptor Grb2
→ allows GTPase RAS to return to inactive phase
Inhibitors that Uncouple the Insulin
Receptor from its Substrate Proteins
SOCS-3 : binds to phosphorylated Tyr960 of IR and prevents
STAT-5B activation by insulin and insulin-induced IRS-1 tyrosine
phosphorylation
Grb10 : interacts with regulatory kinase loop of IR and inhibition
of insulin signaling