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Crk: The First Identified Adaptor Protein

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Crk: The First Identified Adaptor Protein Kathy Abernethy Identification of Crk v-Crk, transforming gene in virally induced chicken tumors, cloned in 1988 v-Crk is ... – PowerPoint PPT presentation

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Title: Crk: The First Identified Adaptor Protein


1
Crk The First Identified Adaptor Protein
  • Kathy Abernethy

2
Identification of Crk
  • v-Crk, transforming gene in virally induced
    chicken tumors, cloned in 1988
  • v-Crk is the oncogene found in CT10 and ASV1
    avian sarcoma viruses
  • Cellular homologs have been implicated in many
    signal transduction pathways, including cell
    differentiation and migration

3
Biochemistry of Crk Protein
  • Crk gene localized to Chromosome 17, specifically
    17p13.3
  • v-Crk protein (p47 gag-Crk) is a fusion product
    of viral gag sequence and cellular Crk protein
  • c-Crk encodes a polypeptide 305 amino acids
    long,of which the first 205 are found in v-Crk
  • This amino acid segment includes an SH2 and SH3
    domain
  • While c-Crk contains 2 SH3 domains, v-Crk has
    only 1

4
  • SH2 and SH3 domains allow Crk to function as an
    adaptor protein. The protein has no kinase
    activity
  • When tyrosine-phosphorylated, adaptor proteins
    are responsible for bringing signal transduction
    components together and facilitating downstream
    signaling
  • Amino-terminal sequences that may be involved in
    c-Crk regulation are absent in v-Crk. c-Crk is
    phosphorylated on Tyr-222 upon cell adhesion,
    negatively regulating SH2/SH3 binding.
    Intermolecular interaction with SH2 domain may be
    source of regulation. Such regulation has not
    been observed in v-Crk.
  • The oncogene also contains amino acid
    substitutions

5
What Proteins Bind to Crk?
  • SH2 domain binds tyrosine-phosphorylated
    proteins
  • 1. FAK-activated Paxillin (a cytoskeletal
    protein)
  • 2. p130Cas, a docking protein that may serve
    as
  • a meeting point for focal adhesions and
    downstream
  • signaling partners
  • 3. Activated Receptor Tyrosine Kinases
    (including PDGF receptor and HEK2)
  • 4. c-Cbl, a docking protein phosphorylated in
    hematopoietic cells. Crk/c-Cbl complexes have
    been seen in Chronic Myelogenous Leukemia (CML)
    cells
  • 5. Insulin Receptor Substrate (IRS-1)

6
What Proteins Bind to Crk?
  • SH3 domain binds proline-rich motifs
  • 1. Abl family kinases
  • 2. PI3 kinase, which acts downstream of
    many RTKs, integrins, and various
    oncogenes
  • 3. KHS and HPK1 kinases
  • 4. Guanine Nucleotide Release Proteins
    (GNRPs), such as C3G
  • 5. Small GTPases (Ras, Rap 1)

7
Crk brings two signaling molecules in close
proximity
8
There are three cellular homologs of Crk
  • Crk-I
  • Crk-II
  • Crk-like (CrkL)
  • Crk-I and Crk-II isoforms formed by alternative
    splicing. Crk-I lacks the last 100 residues of
    Crk-II
  • Crk-type proteins found in vertebrates, flies,
    and nematodes
  • Crk proteins observed in both embryos and adults

Nishihara et al.
9
Crk Expression in Normal Human Tissue Based on
DNA Array

In addition, Northern Blot Analysis shows c-Crk
mRNA in every tissue and organ, suggesting that
the protein is a common signal transduction
molecule
10
What Happens to Cells Lacking Crk?
  • Imaizumi et al. generated mutant Crk mice by
    inserting trap vector into c-Crk gene. A
    truncated Crk protein was expressed, containing 1
    SH2 and 1 SH3 domain. This structure is similar
    to Crk-I protein, so the insertion was considered
    a Crk-II mutation.
  • Homozygous mutant mice did not show any
    abnormalities
  • Conclusion drawn that Crk-II is not essential for
    embryonic development since Crk-family adaptors
    can substitute for one another in signaling
    cascades

11
What About Other Crk Mutations?
  • As previously mentioned, v-Crk possesses 1 SH3
    domain, while c-Crk contains 2 (N-terminal SH3(1)
    domain and C-terminal SH3(2) domain).
  • Ogawa et al. made mutant Crk mice B-Crk lacked
    SH3(2) domain and D-Crk lacked SH3 (1) domain
  • Cells expressing either B-Crk or v-Crk displayed
    morphological alterations and increased tyrosine
    phosphorylation of proteins (specifically, p130).
    Tyrosine phosphorylation levels were 10-20 times
    higher in B-Crk cells than c-Crk or D-Crk cells
  • These results indicate that the C-terminal SH3
    domain is responsible for negatively regulating
    tyrosine phosphorylation of downstream molecules
  • Both B-Crk and v-Crk lack this C-terminal SH3
    domain, which may contribute to the altered
    appearance and transforming ability through
    elevated tyrosine phosphorylation

12
How is Crk Expressed in Tumors?
  • Nishihara et al. performed PCR, using wild-type
    Crk DNA, on 40 different human tumors. All
    PCR-amplifications were successful, showing that
    Crk protein was not mutated in these tumors.
  • Immunostaining was performed on these same 40
    tumors, using anti-Crk polyclonal antibody.
    Significant levels of Crk were isolated in
    carcinoma of lung, breast, and stomach, as well
    as intrapelvic tumors
  • In control tissues, Crk was detected in ependymal
    cell layer in brain, bronchial epithelium of
    lung, and bile duct epithelium of liver. Crk was
    only detected in normal tissue possessing highly
    proliferative cells.
  • Expression levels of Crk were examined in
    cultured cell lines via immunoblotting. Growth
    rates of the lung cancer cell lines PC-3 and
    NPC-8 were significantly higher than the others,
    and Crk expression levels were higher in these
    cell lines as well. Expression of downstream
    molecules was also significantly elevated in the
    PC-3 and NPC-8 lines.

13
Immunostaining of Various Human Tumors
  • A- Lung cancer
  • B- Gastric cancer
  • C- Breast cancer
  • D- Intrapelvic tumor
  • (Chondrosarcoma)
  • E- Carcinosarcoma
  • F- AAH of lung
  • G- Normal bronchial mucosa
  • H- Normal intrahepatic
  • bile duct
  • Nishihara et al.

14
Growth Rates and Expression Levels of Crk and
DOCK180 (A Downstream Protein Involved in Cell
Motility)
Nishihara et al.
15
In Conclusion
  • Nishihara et al. hypothesized that elevated Crk
    levels may be due to increased promotor activity.
    Crk promotor activity was high in human colon
    cancer and embryonal kidney cell lines, as
    compared to that of other promotors.

16
What Exactly is the Role of Crk in Tumor
Formation?
  • Lamorte et al. injected Crk-1 SH2/SH3 and Crk-I
    SH3 mutants into kidney epithelial cells.
  • Crk-1 SH2/SH3 mutant cells displayed lamellipodia
    formation and spreading in response to hepatocyte
    growth factor (HGF), while Crk-I SH3 mutants
    failed to spread or form lamellipodia.
  • In Crk-I SH2/SH3 mutants, Crk-II and CrkL were
    able to function in place of the non-functional
    protein. However, Crk-1 SH3 mutant competed with
    Crk-II and CrkL, thus preventing signal
    transduction.
  • These findings suggest that Crk proteins must be
    able to couple tyrosine-phosphorylated proteins
    with downstream molecules in order for cell
    spreading to occur.

17
In Addition
  • MDCK kidney epithelial cells injected with CrkII
    or CrkL (mimicking overexpression) displayed
    membrane extensions in the absence of HGF, an
    external growth signal.
  • These cells also displayed an enhanced C3G/CrkII
    association. Western blot analysis showed that
    Rap1-GTP and Rac1-GTP (activated by C3G) levels
    were elevated in MDCK cells overexpressing Crk.
    These 2 proteins are involved in lamellipodia
    formation.

18

Lamorte et al.
19
How Else Does Crk Affect Cell Spreading?
  • Breast cancer epithelial cell lines
    (T47D)overexpressing Crk-II failed to stain for
    beta-catenin, a protein involved in adherens
    junctions. T47D cells stained well for
    beta-catenin.
  • Hence, Crk-II overexpression promotes the loss of
    beta-catenin and contributes to cell dispersal.

20
Lamorte et al.
21
Similarly
  • Uemura and Griffin found that cells
    overexpressing Crk showed 2.8-fold higher
    migration on fibronectin-coated surfaces.
  • Effects of Crk mutations were examined.
    Overexpression of Crk SH2 mutants did not alter
    cell migration (in comparison with control
    cells). Overexpression of Crk SH3(N) mutants
    actually inhibited migration. Crk SH3(C) mutants
    enhanced cell migration, but to lesser extent
    than Crk-overexpressed cells. Crk SH2/SH3(C and
    N) overexpressed mutants reduced cell migration.
  • Results suggest that SH2 and SH3(N) are required
    for enhancement of cell migration.

22

Uemura and Griffin
23
What is the Take-Home Lesson?
  • Crk is an adaptor protein consisting of SH2 and
    SH3 domains
  • Binds many different proteins to form a
    signaling cluster
  • Cause of oncogenic activity is unclear, maybe
    protein overexpression or mutation in regulatory
    domains
  • Crk oncogene believed to enhance cell migration
  • Crk is also involved in cell differentiation and
    is a substrate of Bcr-Abl oncogene (CML)
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