SIGNALING FROM THE CELL SURFACE TO THE NUCLEUS

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					          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                   TGF-b1
SUPERFAMILY INCLUDES
TGF- ß1, TGF- ß2, TGF- ß3,
ACTIVIN, INHIBIN,
MULLERIAN INHIBITING
SUBSTANCE,

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.
                           THE TGFb
                          SIGNALING
TGFb SIGNALS THROUGH
HETEROMERIC
COMPLEXES OF
                          PATHWAY
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
                        TGF-b
                        Type II               Type I
                        Receptors             Receptors
            Cell                          P
            Membrane                P
                                    P
                                          P




                                P

 THE TGFb     Smad 4
                                                  Smad 3

SIGNALING    Nuclear
PATHWAY      Membrane



                             mRNA
                P

                    TFE3                (Gene encoding inhibitor of protease
                                        that degrades extracellular matrix
            PAI-1                       proteins)
            promoter
                                mRNA
                    P


                                         (Gene encoding cell- cycle
                p 15 gene                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
   Anima tion de compressor
are neede d to se e 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           CFU-GM
                                           G-CSF

AND DIFFERENTIATION                                              Granulocyte s

OF ERYTHROID                           IL-3, GM-CSF, SCF
                                       IL-6
PROGENITOR
CELLS TO MATURE                            M-CSF
                             CFU-MEG                              M onoc yte s
RED CELLS
                                       SCF    TPO
                                  IL-3, GM-CSF
               CFU-GEMM                                         Plate le ts
                             BFU-E           CFU-E
                                     Epo
                                     SCF             Epo

                                 GM-CSF
                                 IL-3
                                                                    Erythrocyte s

 Pluripotent                  CFU-Eo
 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 HORMONE-
 INDUCED OR HORMONE-
STABILIZED 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              SHP1   68 kDa CYTOSOLIC PROTEIN
            TYROSINE KINASE                               TYROSINE PHOSPHATASE
          HOMOLOGOUS TO JAK1 AND                         EXPRESSED ONLY IN
            TYK2                                          HEMATOPOIETIC CELLS
          WIDELY EXPRESSED IN                           MUTATED IN Motheaten MICE
            HEMATOPOIETIC CELLS AND
            FIBROBLASTS                                                 CATALYTIC
                                                        SH2      SH2     DOMAIN
          NO SH2 OR SH3 DOMAINS
               PSEUDO-KINASE
  N-TERMINAL       DOMAIN      KINASE DOMAIN




  CONSERVED    MODULATORY      TYROSINE KINASE
                                                          EPO

                          EPO- INDUCED RECEPTOR
                             DIMERIZATION;
                         TRANS-PHOSPHORYLATION
                                 OF JAK2
TWO POSSIBLE
MECHANISMS     JAK 2                       P      JAK 2         JAK 2   P



BY WHICH EPO
 ACTIVATES
   THE EPO
  RECEPTOR
                                                          EPO
                              EPO- INDUCED
                         CONFORMATIONAL CHANGE OF
                            DIMERIC RECEPTOR;
                          TRANS-PHOSPHORYLATION
                                 OF JAK2



               JAK 2   JAK 2               P      JAK 2         JAK 2   P
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 TWO-
PRONGED SOCKET ON
THE SURFACE OF THE SH2
DOMAIN. THE PHOSPHATE
GROUP COVALENTLY
ATTACHED TO THE
TYROSINE RESIDUE
IS LIGHT BLUE.
DIMERIZATION OF STAT PROTEINS LEADS
  TO FORMATION OF A FUNCTIONALLY
   ACTIVE TRANSCRIPTION FACTOR




                 P
                      DIMERIZATION OF
             STAT     STAT PROTEIN
                      BY BINDING OF
                      PHOSPHOTYROSINE
                      TO THE SH2 DOMAIN
                      ON THE PARTNER
                      SUBUNIT


        DIMER OF STAT
        PROTEIN IS
        FUNCTIONAL




                                   P
        TRANSCRIPTION       STAT
        FACTOR: MOVES
        INTO NUCLEUS,
        BINDS TO DNA, AND       STAT
                            P

        ACTIVATES
        TRANSCRIPTION OF
        THE BCL-X ANTI-
        APOPTOTIC PROTEIN
        AMONG OTHERS
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 UBIQUITIN-
TAGGED 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. NF-
kB ENTERS THE NUCLEUS, BINDS TO
SPECIFIC SEQUENCES IN DNA AND
REGULATES TRANSCRIPTION.

				
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