The CrkL Adapter Protein Is Required for Type I Interferon-Dependent Gene Transcription and Activation of the Small G-Protein Rap1

We sought to determine the functional role of the CrkL adapter protein and downstream pathways in interferon signaling. In experiments using CrkL −/− mouse embryonic fibroblasts, we found that CrkL is required for IFNα-dependent gene transcription via GAS elements, apparently via the formation of DNA-binding complexes with Stat5. On the other hand, gene transcription via ISRE elements is intact in the absence of CrkL, indicating that the regulatory effects on gene transcription are mediated only via the formation of CrkL:Stat5 complexes. Our studies also indicate that activation of the small GTPase Rap1 by IFNα is defective in cells lacking CrkL, indicating that the protein plays a critical role in regulating activation of the growth inhibitory C3G/Rap1 pathway. The IFNα-inducible activation of the small GTPase Rap1 requires a functional N-terminus SH3 domain in the CrkL protein, while the C-terminus SH3 domain does not appear to play a role in such a CrkL-function. We also demonstrate that both the Tyk-2 and Jak-1 kinases are required for activation of the CrkL/Rap1 pathway, as the Type I IFN-dependent GTP-bound form of Rap1 is inhibited by overexpression of dominant-negative Tyk-2 or Jak-1 mutants and is defective in cells lacking Tyk-2 or Jak-1. Taken altogether, these findings indicate that CrkL provides an important link between Jak-kinases and downstream cascades that play critical roles in IFN-dependent transcriptional regulation and induction of growth inhibitory responses.