The integration of signals triggered by diverse stimuli is essential for cell communication in higher organisms and is primarily achieved through the assembly of multiprotein signaling nodes. The Crk-associated substrate (Cas) and novel SH2-containing protein (NSP) families form one such class of signaling nodes, and correspondingly harbor multiple protein-protein interaction domains and motifs that mediate association with an abundance of cell signaling factors. This allows them to function as mediators that integrate signals emanating from cell adhesion and environmental stimuli to promote cell migration, adhesion and invasion. As such, disregulation of signaling through Cas and NSP proteins is relevant to a spectrum of disease processes, particularly tumor progression and cancer metastasis.
The complexity of NSP-Cas functions is best exemplified by two prominent family members p130Cas and the NSP protein BCAR3, which are also known for their ability to confer antiestrogen resistance in breast cancer1,2
. Firmly embedded in the Src-Crk signaling axis, p130Cas interacts with more than a dozen signaling factors besides Src family kinases and Crk, including FAK, PYK2, FRNK, RapGEF1, Aurora kinase A, PI3K, NMP4, NCK1 and SHIP2 (reviewed in ref 3
). BCAR3 is associated with enhanced PI3K activity downstream of growth factor receptors and subsequent activation of Rac, Cdc42, AKT and PAK14
. Besides this spectrum of signaling partners, p130Cas and BCAR3 also bind directly to each other5
, thereby linking their respective signaling networks to form a platform that mediates migratory signaling. In addition to BCAR3, p130Cas is also capable of interacting with NSP family members NSP3 and NSP16,7
. On the other hand, BCAR3 has been shown to interact with HEF1 (Cas-L) in addition to p130Cas8
. Further combinations are created by association of the NSP family members NSP3 and NSP1 with the Cas proteins Cas-L and Efs, adding to the diversity of NSP–Cas signaling networks9–11
. NSP and Cas family proteins are therefore capable of propagating class-specific but promiscuous combinatorial networks, which are each essential to allow their diverse functions in specialized cellular processes. As such the effect of aberrant NSP–Cas signaling is manifested in a range of disease processes. p130Cas and BCAR3 have long been known to exert a concerted effect in cancer12,13
, while the NSP3 (SHEP1)–Cas-L signaling node has recently been found to be crucial for B-cell migration and maturation9,11
. Additionally the NSP3–p130Cas module is reported to be essential in neuronal cells in for proper olfactory development, and when absent causes a phenotype reminiscent of the human developmental disorder Kallmann syndrome14
. These examples underline the central importance of distinct NSP–Cas modules in cellular processes that require integration of adhesion and chemotactic stimuli.
While it is known that the respective C-terminal domains of Cas and NSP proteins are fundamental to their interaction (Supplementary Fig. 1a
, it has remained unclear at the molecular level how different family members can interact in this class-specific yet promiscuous manner. Moreover the predicted nature of the C-terminal Cas and NSP domains suggest an unprecedented interaction mechanism. The NSP-binding portion of Cas proteins has been proposed to assume a focal-adhesion targeting (FAT) domain-like fold, while the C-terminal domain of NSP proteins shares weak homology with Cdc25-homology domains (Cdc25-hd) found in GTPase exchange factors15,16
. However, the molecular mechanism of a direct Cdc25-hd:FAT interaction is currently without precedent, and the question of how this influences the putative activities of each domain while mechanically linking NSP and Cas signaling remains outstanding.
To shed light on the molecular features enabling the formation of Cas–NSP signaling modules, we have solved the crystal structures of the C-terminal region of unbound human BCAR3 and of the complex between human NSP3 and p130Cas. These structures in combination with our biochemical and biological analyses show that the Cdc25-homology domain present in NSP proteins has evolved as an adaptor domain based on an enzymatic fold used in RasGEFs. This unique NSP structure is specifically tailored to tightly bind the FAT domain of Cas proteins using a distinctive extended interface. Furthermore, the sophisticated and class-specific interaction patterns observed in the NSP3 and p130Cas interfaces are conserved among the members of both families. As a result, NSP and Cas proteins can promiscuously interact with each other to form various combinations of signaling modules that finely regulate cellular processes.