Title: Crk: The First Identified Adaptor Protein
1Crk The First Identified Adaptor Protein
2Identification 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
3Biochemistry 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
5What 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)
6What 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)
7Crk brings two signaling molecules in close
proximity
8There 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.
9Crk 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
10What 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
11What 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
12How 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.
13Immunostaining 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.
14Growth Rates and Expression Levels of Crk and
DOCK180 (A Downstream Protein Involved in Cell
Motility)
Nishihara et al.
15In 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.
16What 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.
17In 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.
18Lamorte et al.
19How 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.
20Lamorte et al.
21Similarly
- 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
23What 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)