Transcript 9/18 - MIT

SIGNALING FROM THE CELL SURFACE
TO THE NUCLEUS
• PROTEIN KINASE A
• PHOSPHORYLATION AND ACTIVATION OF CREB TRANSCRIPTION FACTOR
• RECEPTOR SERINE KINASES - RECEPTORS
FOR THE TGF-ß SUPERFAMILY
• PHOSPHORYLATION AND ACTIVATION OF SMAD TRANSCRIPTION FACTORS
• PARTNERING WITH OTHER TRANSCRIPTION FACTORS TO ACTIVATE
TRANSCRIPTION OF SPECIFIC GENES
• PROTEIN- TYROSINE KINASE RECEPTORS
• RECEPTORS LINKED TO PROTEIN- TYROSINE
KINASES - THE CYTOKINE RECEPTOR SUPERFAMILY
• RECEPTORS LINKED TO PROTEOSOME- MEDIATED
DEGRADATION OF INHIBITORS OF SPECIFIC
TRANSCRIPTION FACTORS
SIGNALING PATHWAYS LEADING TO
ACTIVATION OF TRANSCRIPTION FACTORS
AND MODULATION OF GENE EXPRESSION
FOLLOWING LIGAND BINDING TO CERTAIN GS
PROTEIN–LINKED RECEPTORS
TGF-b: Key Roles in Controlling
Cell Proliferation and Synthesis of the
Extracellular Matrix
THE TGF- ß
SUPERFAMILY INCLUDES
TGF- ß1, TGF- ß2, TGF- ß3,
ACTIVIN, INHIBIN,
MULLERIAN INHIBITING
SUBSTANCE,
TGF-b1
AND AT LEAST 16
BONE MORPHOGENETIC
PROTEINS
Hinck et al., (1996) Biochemistry
TGF-b: Key Roles in Controlling
Cell Proliferation and Synthesis of the
Extracellular Matrix
BIOLOGICAL FUNCTIONS OF TGF-ß INCLUDE:
• INHIBITION OF CELL PROLIFERATION
• INDUCES INHIBITORS OF CYCLIN - DEPENDENT KINASES
• TYPE II RECEPTOR FREQUENTLY LOST OR MUTATED IN CANCERS
• INDUCTION OF SYNTHESIS OF EXTRACELLULAR MATRIX PROTEINS:
FIBRONECTIN, COLLAGENS, PROTEOGLYCANS
• INHIBITION OF SYNTHESIS OF EXTRACELLULAR PROTEASES:
COLLAGENASE, PLASMINOGEN ACTIVATOR
• INDUCTION OF SYNTHESIS OF INHIBITORS OF
EXTRACELLULAR PROTEASES
• PROMOTION OF CELL MATRIX AND CELL- CELL ATTACHMENT
Schematic diagram of formation of mature
dimeric TGFb proteins from secreted
monomeric TGFb precursors.
TGFb SIGNALS THROUGH
HETEROMERIC
COMPLEXES OF
TYPES I AND II
SERINE/THREONINE
KINASE RECEPTORS,
LEADING TO
PHOSPHORYLATION OF
EITHER SMAD2 OR SMAD3.
A COMPLEX OF ONE
OF THESE PHOSPHORYLATED
SMAD PROTEINS AND SMAD4
THEN TRANSLOCATES
TO THE NUCLEUS,
WHERE IT BINDS
TO OTHER TRANSCRIPTION
FACTORS TO ACTIVATE
TRANSCRIPTION
OF A VARIETY OF GENES
THE TGFb
SIGNALING
PATHWAY
TGF-b
Type I
Receptors
Type II
Receptors
Cell
Membrane
THE TGFb
SIGNALING
PATHWAY
P
P
P
P
P
Sm ad 3
Sm ad 4
Nuclear
Membrane
mRNA
P
TFE3
PAI-1
promoter
P
p 15 gene
(Gene encoding inhibitor of protease
that degrades extracellular m atrix
proteins)
mRNA
(Gene encoding cell- cycle
inhibitor)
unknown transcription factors
COMBINATORIAL ACTIVATION OF
TRANSCRIPTION BY SMAD PROTEINS
SMAD3 PROTEINS BIND ONLY TO 4 BASE PAIRS OF DNA: 5’ AGAC 3’
EACH TFE3 TRANSCRIPTION FACTOR BINDS TO A 3 BASE PAIR SEQUENCE 5’ CAC 3’
A DIMER OF TWO TFE3s BINDS TO A 6 BASE PAIR SEQUENCE 5’ CACGTG 3’ (GTG IS
THE COMPLEMENT OF CAC)
THUS A SEQUENCE 5’ AGACxxxCACGTG 3’ BINDS ONE SMAD3 PROTEIN AND ONE
TFE3 DIMER IN A PRECISE ARRANGEMENT, ALLOWING FOR TRANSCRIPTION
ACTIVATION, IN THIS CASE OF THE PAI-1 GENE.
QuickTime™ and a
Animation decompressor
are needed to see this picture.
Role of TGF-b in Human Cancer
Human Diseases with Alterations
in the TGF-b Signaling Pathway
SIGNALING FROM THE CELL SURFACE
TO THE NUCLEUS
• RECEPTORS LINKED TO PROTEIN- TYROSINE
KINASES - THE CYTOKINE RECEPTOR SUPERFAMILY
• PHOSPHOTYROSINE RESIDUES BINDING TO SPECIFIC SH2 DOMAINS
• ACTIVATION OF STAT TRANSCRIPTION FACTORS
• PARTNERING OF STATs WITH OTHER TRANSCRIPTION FACTORS
• TERMINATION OF SIGNALING BY ACTIVATION OF PROTEIN
TYROSINE PHOSPHATASES
• INHIBTION OF SIGNALING BY PROTEINS CONTAINING ONLY SH2 DOMAINS
• RECEPTORS LINKED TO PROTEOSOME- MEDIATED
DEGRADATION OF INHIBITORS OF
CERTAIN TRANSCRIPTION FACTORS
HEMATOPOIESIS
EPO ACTS TO STIMULATE
THE PROLIFERATION
AND DIFFERENTIATION
OF ERYTHROID
PROGENITOR
CELLS TO MATURE
RED CELLS
G-CSF
CFU-GM
Granulocytes
IL-3, GM-CSF, SCF
IL-6
M -CSF
CFU-MEG
SCF
Monocytes
TPO
IL-3, GM-CSF
CFU-GEMM
Platelets
BFU-E Epo
CFU-E
SCF
Epo
GM -CSF
IL-3
Erythrocytes
CFU-Eo
Pluripotent
Stem Cell
IL-3, GM-CSF
Eosinophils
Lymphoid
Progenitor
ERYTHROPOIETIN (EPO)
THE PROTEIN THAT CONTROLS
RED BLOOD CELL PRODUCTION
165 AMINO ACIDS
~ 40% CARBOHYDRATE
• PRODUCED BY THE KIDNEY IN RESPONSE TO LOW O 2 PRESSURE IN THE
BLOOD
• BINDS TO EPO RECEPTORS ON THE SURFACE OF ERYTHROCYTE
PROGENITOR CELLS IN THE BONE MARROW
• STIMULATES THESE CELLS TO DIVIDE 5 TO 7 TIMES; EACH OF THE ~30
TO 100 DAUGHTERS THEN DIFFERENTIATES INTO A RED BLOOD CELL
• USED CLINICALLY TO TREAT ANEMIA CAUSED BY KIDNEY FAILURE OR
BY DISEASES SUCH AS AIDS
EPO REGULATES RED CELL MASS IN RESPONSE TO TISSUE HYPOXIA
RED CELL MASS
Tissue pO2
+
Epo
EPO “GENE KNOCK- OUT” MICE ARE NORMAL
EXCEPT THEY HAVE NO ADULT- TYPE RED
BLOOD CELLS AND DIE AT EMBRYONIC DAY 14
CYTOKINE RECEPTOR SUPERFAMILY
TWO CONSERVED
DISULFIDE BONDS
CONSERVED
Trp- Ser- Xaa- Trp -Ser
SEQUENCE
Extracellular
Intracellular
BOX 1
BINDING SITE FOR JAK
NO ENZYMIC ACTIVITY
IN CYTOSOLIC DOMAIN
CYTOKINE RECEPTORS
THAT FORM HORMONEINDUCED OR HORMONESTABILIZED HOMO-DIMERS:
• ERYTHROPOIETIN (EPO)
• GRANULOCYTE COLONY
STIMULATING FACTOR
• THROMBOPOIETIN (TPO)
• PROLACTIN (PRL)
• GROWTH HORMONE (GH)
SALES OF EPO AND G-CSF ARE IN
EXCESS OF $4 AND $2 BILLION/
YEAR, RESPECTIVELY
STRUCTURE
OF HUMAN
GROWTH
HORMONE
LIKE EPO AND OTHER
CYTIOKINES, GROWTH
HORMONE FORMS A 4- ALPHA
HELIX BUNDLE.
AMINO ACIDS THAT BIND TO
THE FIRST GROWTH
HORMONE RECEPTOR ARE IN
GREEN; THOSE THAT BIND
TO THE SECOND GROWTH
HORMONE RECEPTOR ARE IN
BLUE
STRUCTURE OF THE
EXTERNAL SEGMENT OF
THE HUMAN GROWTH
HORMONE RECEPTOR
THE PLASMA MEMBRANE IS AT
THE BOTTOM OF THE FIGURE
AMINO ACIDS THAT BIND
GROWTH HORMONE ARE IN BLUE
AMINO ACIDS THAT BIND THE
SECOND MOLECULE OF
GROWTH HORMONE RECEPTOR
ARE IN GREEN
THREE- DIMENSIONAL STRUCTURE OF THE COMPLEX
OF ONE MOLECULE OF HUMAN GROWTH HORMONE
AND TWO GROWTH HORMONE RECEPTORS
PLASMA MEMBRANE
IS AT THE BOTTOM
OF THE FIGURE
SIGNAL TRANSDUCTION PROTEINS THAT
BIND TO THE CYTOSOLIC DOMAIN
OF THE ERYTHROPOIETIN RECEPTOR
JAK2
130 kDa CYTOSOLIC PROTEIN
TYROSINE KINASE
HOMOLOGOUS TO JAK1 AND
TYK2
WIDELY EXPRESSED IN
HEMATOPOIETIC CELLS AND
FIBROBLASTS
NO SH2 OR SH3 DOMAINS
N-T ERMINAL
CONSERVED
PSEUDO-KINASE
DOMAIN
MODULATORY
KINASE DOMAIN
T YROSINE KINASE
SHP1
68 kDa CYTOSOLIC PROTEIN
TYROSINE PHOSPHATASE
EXPRESSED ONLY IN
HEMATOPOIETIC CELLS
MUTATED IN Motheaten MICE
SH2
SH2
CAT ALYTIC
DOMAIN
EPO
TWO POSSIBLE
MECHANISMS
BY WHICH EPO
ACTIVATES
THE EPO
RECEPTOR
EPO- INDUCED RECEPTOR
DIMERIZATION;
TRANS-PHOSPHORYLATION
OF JAK2
JAK 2
P
JAK 2
EPO- INDUCED
CONFORMATIONAL CHANGE OF
DIMERIC RECEPTOR;
TRANS-PHOSPHORYLATION
OF JAK2
JAK 2
JAK 2
P
JAK 2
JAK 2
P
JAK 2
P
EPO
SIGNAL TRANSDUCTION BY THE EPO RECEPTOR
ACTIVATED JAK2 PHOSPHORYLATES UP TO 8
TYROSINE RESIDUES ON THE CYTOSOLOC DOMAIN
OF THE EPO RECEPTOR. EACH PHOSPHOTYROSINE
CAN FORM THE “DOCKING SITE” FOR THE SH2
DOMAIN OF A SIGNAL TRANSDUCTION PROTEIN
MODEL OF AN SH2 DOMAIN BOUND TO A
SHORT TARGET PEPTIDE.
IN THIS TARGET PEPTIDE,
THE PHOSPHOTYROSINE
(P-TYR) AND ISOLEUCINE
(+3ILE) FIT INTO A TWOPRONGED SOCKET ON
THE SURFACE OF THE SH2
DOMAIN. THE PHOSPHATE
GROUP COVALENTLY
ATTACHED TO THE
TYROSINE RESIDUE
IS LIGHT BLUE.
P
DIMERIZATION OF STAT PROTEINS LEADS
TO FORMATION OF A FUNCTIONALLY
ACTIVE TRANSCRIPTION FACTOR
P
DIMER OF STAT
PROTEIN IS
FUNCTIONAL
TRANSCRIPTION
FACTOR: MOVES
INTO NUCLEUS,
BINDS TO DNA, AND
ACTIVATES
TRANSCRIPTION OF
THE BCL-X ANTIAPOPTOTIC PROTEIN
AMONG OTHERS
P
DIMERIZATION OF
STAT PROTEIN
BY BINDING OF
PHOSPHOTYROSINE
TO THE SH2 DOMAIN
ON THE PARTNER
SUBUNIT
STAT
STAT
STAT
TERMINATION OF SIGNAL
TRANSDUCTION
BY THE EPO RECEPTOR
TERMINATION OF SIGNAL TRANSDUCTION
BY THE EPO RECEPTOR #2
GENERAL STRUCTURE AND
ACTIVATION OF RECEPTOR
TYROSINE KINASES (RTKS)
AS WITH THE EPO RECEPTOR,
LIGAND BINDING INDUCES A
CONFORMATIONAL CHANGE
THAT PROMOTES OR STABILIZES
RECEPTOR DIMERS.
THE KINASE ACTIVITY OF EACH
SUBUNIT OF THE DIMERIC
RECEPTOR INITIALLY
PHOSPHORYLATES TYROSINE
RESIDUES NEAR THE CATALYTIC
SITE IN THE OTHER SUBUNIT,
CAUSING ITS ACTIVATION.
SUBSEQUENTLY, TYROSINE
RESIDUES IN OTHER PARTS OF THE
CYTOSOLIC DOMAIN BECOME
PHOSPHORYLATED AND SERVE
AS DOCKING SITES FOR SH2
DOMAINS OF SIGNALING PROTEINS
Structure of the FGF FGR Receptor Complex
Structures of MAP kinase in its inactive,
unphosphorylated form and active,
phosphorylated form
Phosphorylation of MAP kinase by MEK at tyrosine 185 (pY185) and threonine 183
(pT183) leads to a marked conformational change in the phosphorylation lip (red).
Cycling of the Ras protein between the inactive form
with bound GDP and the active form with bound GTP
Activation of Ras
following binding
of a ligand to a
RTK
Model of SH3 domain bound to a short target peptide
Kinase cascade
that transmits
signals
downstream from
activated Ras
protein
Signaling pathways leading to activation of
transcription factors and modulation of gene
expression following ligand binding to RTKs
Activation of
protein kinase B
by the PI- 3’
kinase signaling
pathway
(part 1).
Activation of protein kinase B by the PI- 3’
kinase signaling pathway (part 2).
Cleavage of PIP2 by phospholipase C (PLC)
yields DAG and IP3.
Elevation of cytosolic Ca2+ via the inositol-lipid
signaling pathway
UBIQUITIN-MEDIATED PATHWAY
FOR DEGRADATION OF
CELLULAR PROTEINS
A CONJUGATING ENZYME
CATALYZES FORMATION
OF A PEPTIDE BOND
BETWEEN THE SMALL
PROTEIN UBIQUITIN (UB)
AND THE SIDE-CHAIN –NH2
OF A LYSINE RESIDUE IN
A TARGET PROTEIN.
ADDITIONAL UB MOLECULES
ARE ADDED, FORMING A
MULTIUBIQUITIN CHAIN.
THIS CHAIN DIRECTS THE
TAGGED PROTEIN TO A
PROTEASOME, WHICH
CLEAVES THE PROTEIN INTO
NUMEROUS SMALL PEPTIDE
FRAGMENTS.
PROTEOLYSIS OF UBIQUITINTAGGED PROTEINS OCCURS
ALONG THE INNER WALL
OF THE CORE.
ACTIVATION OF THE
TRANSCRIPTION
FACTOR NF-kB
MANY DIFFERENT EXTRACELLULAR
SIGNALS CAN INDUCE ACTIVATION OF
NF-kB; THESE SIGNALS ACTIVATE AN I-kB
KINASE COMPLEX.
THIS COMPLEX PHOSPHORYLATES TWO
N-TERMINAL SERINE RESIDUES IN I-kB.
PHOSPHORYLATED I-kB IS
UBIQUITINATED AND SUBSEQUENTLY
DEGRADED BY THE PROTEOSOME.
REMOVAL OF I-kB UNMASKS THE
NUCLEAR LOCALIZATION SITES IN BOTH
THE P50 AND P65 SUBUNITS OF NF-kB. NFkB ENTERS THE NUCLEUS, BINDS TO
SPECIFIC SEQUENCES IN DNA AND
REGULATES TRANSCRIPTION.