Protein Tyr Kinases and Phospha

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 Protein Tyr Kinases and Phospha Powered By Docstoc
					Biochem 503            November 25, 2008


    Protein Tyr Kinases

Assigned reading (pdf are on-line)

•   Tony Pawson (2004) Cell 116: 196-203
•   Joseph Schlessinger (2002) Cell 110: 660-672
•   Blume-Jensen & Hunter (2001) Nature 411:355-365
Protein Kinases in the Human Genome
              Protein Tyr Kinases
                                   Cell Signaling
                                   Technologies
                                <www.cellsignal.com>




                        Protein Ser/Thr Kinases
Protein Tyr Kinases - two
         classes

                                           “Receptor”
                                           i.e.
                                           transmembrane
              QuickTime™ and a
     TIFF (Uncompressed) decompressor
        are need ed to see this picture.
1. History of Protein Tyr Phosphorylation
                                                     read assigned Pawson article in Cell, 2004


               A. Viral oncogenes; SRC and Cancer                                                           Quic kT ime™ and a
                                                                                                  T IF F (Unc om pres sed) dec ompres s or
                                                                                                     are needed to see this pict ure.




                                     1917            Peyton Rous - avian sarcoma virus,

                                     1960’s the src virus gene as causative agent                 Peyton Rous


                                     1976            Varmus & Bishop show a c-src gene in humans

                                                     oncogene and proto-oncogene
                  Qu i ckTi me ™ an d a
                                                            - viral and endogenous
       TIFF (Un co mp re ss e d) de co mp re ss or
          a re ne ed ed to se e thi s p i ctu re .



                                                               the virus as an agent to distort
                                                               endogenous signaling that controls cell
J. Michael Bishop and Harold Varmus                            proliferation
UCSF; Nobel Prize
1978-1980                                          Ray Erikson (Univ. of Colorado) with postdoc Joan Brugge
                                                       (now both at Harvard)

                                                     antisera from rabbits injected with RSV                     QuickT ime™ and a


                                                     IgG against the src protein: p60src
                                                                                                      T IF F (Uncompressed) decom pressor
                    c             n
                 Qu i k Ti me ™ a d a                                                                     are needed to see this pict ure.
     F             re      d             e
  TIF (Un co m p s s e ) d e co m p r s s o    r
                 e            t      c
     a re n e e d d to s e e h i s p i tu re .




                                                     Marc Collett and Erikson
                                                     src protein immunoppt. + ATP-32P, get 32P-IgG HC (55 kDa)

                             There’s a kinase activity in v-src!

                                                     Tony Hunter (Salk Inst., LaJolla CA)
                QuickTime™ and a
     TIF F (U ncompr essed) decompr essor
        are needed to see this pictur e.
                                                     recognizes P-Tyr in ppt of PYmTAg using 2D TLE method
                                                     kinase activity, but unlike any known at the time.

                                                               HN                              O
                                                                 CH-CH2               O        P--O
                                                           -                                   O
                                                               O=C             Phospho-Tyrosine
                                                                 NH
                                                                                                          B. growth factor receptors
    Stanley Cohen                                                                                         1980      EGF receptor, EGF found as hormone
                                                                 QuickT ime™ and a
    Vanderbilt                                         T IF F (Uncompressed) decompressor
                                                          are needed to see this picture.


    (Nobel Prize)                                                                                                   action of EGF in A431 cells
                                                                                                                    found 32P-labeling of 150 kDa band
                                                                                                                    mistaken as P-Thr, then shown as P-Tyr

                                                   Ora Rosen                                              1982      insulin receptor kinase activity
                                                   Einstein Med. Col                                                Tyr auto-phosphorylation-multiple P-Tyr
                                                   deceased 1990                                                    sequence reveals pre-pro-protein,cleaved
                                                                                                                    and dimerized into a2b2

                                                                                                          1983-85     technology of gas-phase peptide
Jos. Schlessinger                                                        Qu i ckTi m e™ a nd a
                                                             TIFF (Un co mp res se d) de c omp res so r                          micro sequencing
                                                                a re ne ed ed to se e th is pi c tu re.

Tel Aviv, NYU, Yale                                                                                                 other GFRs shown to have PTK
                                                                                                                            activity: IGF-1, PDGF, FGF

                                                                                                          v-erbB oncogene is cytoplasmic domain
                Q uic kT im e™ a n d a
    T IFF ( Un co m pr e ss e d) d ec o mp r es s or     Tony Pawson                                      of EGF receptor…..virus produces unregulated
        a re n ee d ed to s e e th is p ictu r e.

                                                         Toronto                                          version of growth regulator enzyme (PTK)

                                                                                                          GRB = growth factor binding proteins……contain
                                                                                                          SH2 domains that bind to P-Tyr
2. Analysis of Protein Phosphorylation
                                                                   Qu i ckTi me ™ an d a
A. P-Tyr vs. P-Ser or P-Thr                 2D method   TIFF (Un co mp re ss e d) de co mp re ss or
                                                           a re ne ed ed to se e thi s p i ctu re .




       a. requires 32P-phosphoamino acid analysis, done by HCl hydrolysis
       and HV-TLE in 2 dimensions, pH 1.9 & pH 3.5, now 1D at pH 2.5.
                Non-quantitative due to different rates of hydrolysis
       b. phospho-specific antibodies P-Tyr (4G10, PY20),
       c. site-specific P-Tyr antibodies                                                        Quic kT ime™ and a
                                                                                      T IF F (Unc om press ed) dec om pres sor
                                                                                         are needed t o see t his pict ure.

                (quantitation but no stoichiometry)      1D method

B. Distribution of P-Tyr
       a. species - metazoan enzymes…. only in multicellular organisms
               P-Tyr in yeast, but no PTKs,
               all P-Tyr from dual specificity kinases
       b. subcellular -membranes >90%, in particulate cell fraction (B above),
               mostly receptors + cytoskeleton or signaling complexes
       c. P-Tyr proteins at UVA led to FAK (Parsons) and MAPK (Weber)
               v-src targets in cells are in signaling pathways
3. Protein Tyr Kinases
A. Receptor Protein Tyr Kinases -(prototype EGFR)
  1. Common Features
      a. single pass transmembrane domain (alpha-helix)
      b. single kinase domain - with or without 'insert’ (docking site)
      c. variable and often large extracellular domain (hormone binding)
      d. small but important juxta-(near) membrane segment (docking)
      e. similar mechanism of activation (dimerization + phosphorylation)

 2. Diversity of cell-surface proteins
      a. many separate genes, wide repertoire (kinase and docking specificity)
      b. complex extracellular domains, Ig and FNII domains
      c. cell adhesion contacts-bidirectional signaling seen in Eph family
Receptor Protein Tyr Kinases - resembling EGFR
3. Activation Mechanism for Receptor PTKs

     a. ligand-induced dimerization in membrane
     b. Tyr phosphorylation of P-loop residues -->activity
     c. phosphorylation of juxtamembrane and C terminal regions
     d. recruitment of substrates and effectors
              SH2 and PTB domains interact with P-Tyr
     e. phosphorylation of recruited substrates: PI3K, PLCg IRS
     f. internalization and other actions (nuclear)
Activation Mechanism for Receptor PTKs




     Assignment: Explain how this model is not accurate,
    relative to new structural data (see Schlessenger paper)
Mechanism for Receptor PTKs Downregulation




      EGFR1 internalized, ErbB2 is not.
      EGF induces degradation, TNFa gives recycling.
Inhibitors of Receptor PTKs in Clinical Use


      Cetuximab
      (Erbitux)




              Gefitinib (Iressa)
              Erlotinib (Tarceva)
Chemical Inhibitors of EGF Receptor Tyr Kinase
Currently in Use in Clinical Oncology - Oral Agents



                           Gefitinib = Iressa = ZD1839




                           Erlotinib = Tarceva = OSI774



                              Lapatinib = GW572016
Mutations in ATP binding site of EGF-R and ERB-
                       B2
  occur in tumors and produces resistance to
                   inhibitors




                               QuickTime™ and a
                      TIFF (Uncompressed) decompressor
                        are neede d to see this picture.




              Int. J. Cancer (2005) 118: 257-262
B. Non-Receptor Protein Tyr Kinases (PTKs)
1. Common Features- prototype is the src family
      a. kinase domain, (SH1) often near C terminus
      b. targeting domains, most often SH2 [P-Tyr] and SH3 [Pro-XX-Pro]
      c. auto-inhibited by multiple mechanisms (see below)
      d. often lipid modified to localize at membrane;
               palmitate, myristate


2. Diversity and Specificity
      a. different tissue and developmental expression
      b. different targeting because all SH3 and SH2 not equivalent
      c. FERM domains send some PTKs to integrins
      d. active site specificity revealed by selective inhibitors
Non-Receptor Protein Tyr Kinases (PTKs)
Development of Chronic Myeloid Leukemia

                         Chromosome
                        re-arrangement

                         produces an
                           activated
                           KINASE
                          BCL-ABL

                          to drive cell
                         proliferation.
Sussessful Targeted Therapy
Gleevec Cures CML Chronic Myeloid Leukemia




                     ATP



                    GleevecTM



BCR-ABL kinase
3. Activation Mechanisms for src-family PTKs

a. covalent - phosphorylation in P-loop; intrasteric effects
      1. phosphorylation of P-loop to allow peptide substrate binding
              AND
      2. dephosphorylation of P-Tyr to relieve intrasteric P-Tyr::SH2

b. allosteric - constraints on C helix and ATP site
      1. SH3 engagement to release conformational restraint
      2. SH2 binding to compete away intrasteric interaction
      Structure of src-family PTKs




Explain src activation using this diagram (next slide)
Activation Mechanisms for src-family PTKs
Orientation of the C Helix is Critical for Kinase
                    Activity
    positions the essential Glu91 for catalysis
C.   Dual-specificity Kinases [Tyr + Ser/Thr]

     1. CDK-kinases that inactivate CDK (Myt1)
     a. dual specificity for Tyr-Thr in CDK N terminal domain
     b. regulators that prevent cell cycle progression
     c. conserved in early eucaryotes…source of P-Tyr where no PTKs

     2. MAP kinase kinases (MEK, for MAP/ERK kinase)
     a. dual specificity for Tyr-X-Thr in activation loop, activate
               substrate kinase
     b. specificity for target kinase, MAPK, JNK, p38K, etc.
     c. inhibited by non-competitive compounds…
               ..novel structure and action and specificity
Structure of Wee1 Kinase Complexed with Active Site
                     Inhibitor




                                QuickTime™ and a
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                         are neede d to see this picture.




    Wee1 looks like a Ser/Thr kinase (in 1o and 3o structures)
            but phosphorylates only Y15 in CDK2.
Structure of Dual-Specificity MEK1 Kinase
with MgATP plus Non-Competitive Inhibitor




                       QuickTime™ an d a
              TIFF (Uncompressed) decompressor
                 are need ed to see this picture .
MEK1 Kinase: Non-competitive Inhibitor Produces
 “Closed” Conformation with Displaced C Helix



                                                   Comparison-Overlap of
                                                   PKA and MEK1
                                                   As a result of the change in
                                                   position of helix C, the side
                                                   chain of the highly conserved
                    QuickTime™ an d a
           TIFF (Uncompressed) decompressor
                                                   glutamate residue Glu114
              are need ed to see this p icture .
                                                   (equivalent to Glu91 in PKA)
                                                   is shifted away from the
                                                   active site and is unable to
                                                   form a critical ion pair with
                                                   the conserved catalytic Lys97
                                                   (equivalent to Lys72 in PKA).

				
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