Transcript Crk: The First Identified Adaptor Protein

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 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
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
 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
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)
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)
Crk brings two signaling molecules in close
proximity…
An example of a signaling complex –
a protein relay system using
phosphorylation as a signal
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
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Nishihara et al.
Crk Expression in Normal Human
Tissue Based on DNA Array
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In addition, Northern Blot
Analysis shows c-Crk mRNA
in every tissue and organ,
suggesting that the protein is
a common signal
transduction molecule
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
What About Other Crk Mutations?
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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 DCrk 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
How is Crk Expressed in Tumors?
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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.
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.
Growth Rates and Expression Levels of Crk and
DOCK180 (A Downstream Protein Involved in
Cell Motility)
Nishihara et al.
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.
What Exactly is the Role of Crk in Tumor
Formation?
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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 tyrosinephosphorylated proteins with downstream molecules in order for cell
spreading to occur.
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.
Lamorte et al.
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.
Lamorte et al.
Similarly…
 Uemura and Griffin found that cells overexpressing Crk showed 2.8fold 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.
Uemura
and
Griffin
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)