Signaling adaptor protein Crk is involved in malignant feature of pancreatic cancer associated with phosphorylation of c-Met.

Signaling adaptor protein Crk has been shown to play an important role in various human cancers. Crk links tyrosine kinases and guanine nucleotide exchange factors (GEFs) such as C3G and Dock180 to activate small G-proteins Rap and Rac, respectively. In pancreatic cancer, various molecular targeted therapies have provided no significant therapeutic benefit for the patients so far due to constitutive activation of KRAS by frequent KRAS mutation. Therefore, the establishment of novel molecular targeted therapy in KRAS-independent manner is required. Here, we investigated a potential of Crk as a therapeutic target in pancreatic cancer. Immunohistochemistry on human pancreatic cancer specimens revealed that the patients with high expression of Crk had a worse prognosis than those with low expression. We established Crk-knockdown pancreatic cancer cells by siRNA using PANC-1, AsPC-1, and MIA PaCa-2 cells, which showed decreased cell proliferation, invasion, and adhesion. In Crk-knockdown pancreatic cancer cells, the decrease of c-Met phosphorylation was observed. In the orthotopic xenograft model, Crk depletion prolonged survival of mice significantly. Thus, signaling adaptor protein Crk is involved in malignant potential of pancreatic cancer associated with decrease of c-Met phosphorylation, and Crk can be considered to be a potential therapeutic molecular target.

ACAP4 interacts with CrkII to promote the recycling of integrin ß1.

ACAP4, a GTPase-activating protein (GAP) for the ADP-ribosylation factor 6 (ARF6), plays import roles in cell migration, cell polarity, vesicle trafficking and tumorigenesis. Similarly, the ubiquitously expressed adaptor protein CrkII functions in a wide range of cellular activities, including cell proliferation, T cell adhesion and activation, tumorigenesis, and bacterial pathogenesis. Here, we demonstrate that ACAP4 physically interacts with CrkII. Biochemical experiments revealed that ACAP4550-660 and the SH3N domain of CrkII are responsible for the interaction. Functional characterization showed that the interaction is required for the recruitment of ACAP4 to the plasma membrane where ACAP4 functions to regulate the recycling of the signal transducer integrin ß1. Thus, we suggest that the CrkII-ACAP4 complex may be involved in regulation of cell adhesion.

Adaptor protein CRK induces epithelial-mesenchymal transition and metastasis of bladder cancer cells through HGF/c-Met feedback loop.

We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial-mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial-mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues.

Temperature Dependence of CN and SCN IR Absorptions Facilitates Their Interpretation and Use as Probes of Proteins.

Cyano and thiocyano groups have received attention as IR probes of local protein electrostatics or solvation, due to their strong absorptions and the ability to site specifically incorporate them within proteins. However, interpreting their spectra requires knowing whether they engage in hydrogen bonds (H-bonds). Existing methods for the detection of such H-bonding interactions are based on structural analysis or correlations between IR and NMR signals and are labor intensive and possibly ambiguous. Here, using model systems we show that the absorption frequency of both probes is linearly correlated with temperature and that the slope of the resulting line (frequency-temperature line slope or FTLS) reflects the nature of the probe's microenvironment, including whether or not the probe is engaged in H-bonds. We then show that the same linear dependence is observed with p-cyano phenylalanine, cyanylated cysteine, or cyanylated homocysteine incorporated at different positions within the N-terminal Src homology 3 domain of the murine adapter protein Crk-II. The FTLSs indicate that p-cyano phenylalanine incorporated at two positions is engaged in strong H-bonding, while it is involved in weaker H-bonding at a third position. In contrast, the FTLS of the cyanylated cysteine or cyanylated homocysteine absorptions indicates that they do not engage in H-bonding at either a buried or surface exposed position. While the differences likely reflect side chain flexibility and the probe's ability to avoid solvent, the data suggest that the temperature dependence of the absorption provides a simple method to gauge the probe's environment, including the presence of H-bonding.