Crk: The First Identified Adaptor Protein

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Crk The First Identified Adaptor Protein

• Kathy Abernethy

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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

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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

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• 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

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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)

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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)

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Crk brings two signaling molecules in close
proximity
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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.
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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
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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

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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

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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.

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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.

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Growth Rates and Expression Levels of Crk and
DOCK180 (A Downstream Protein Involved in Cell
Motility)
Nishihara et al.
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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.

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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.

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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.

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Lamorte et al.
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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.

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Lamorte et al.
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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.

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Uemura and Griffin
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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)