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 Nishiharaet 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)