Crk and CrkL adaptor proteins: networks for physiological and pathological signaling

Abstract

The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses.

Figure 1

(A). Coupling of signals through modular SH2 and SH3 domains. Signals are initiated via extracellular factors that induce intracellular tyrosine phosphorylation (indicated by the light switch) which are subsequently relayed to downstream targets through SH3 binding partners (indicated by arrowhead). (B). Structure of the Crk family of proteins. The domains are boxed: SH2, Src homology 2; SH3, Src homology 3; Gag, viral group specific antigen; Y221 or Y207, negative regulatory phosphorylation site. The structure of Src is shown at the top of the figure to indicate its spatial arrangements compared to Crk. TK, tyrosine kinase domain.

Figure 2

Structural characteristics and interacting proteins of p130cas. (A). p130Cas is a nonezymatic scaffolding protein that contains, (i) an N-terminal SH3 domain that binds FAK and Pyk2, 15 repeats of a YxxP motif, a serine-rich motif that binds Src kinases, and a conserved C-terminal region that binds members of the Chat family of proteins. (B). Signal transduction by the p130Cas scaffold protein. The central substrate region of p130Cas (shown in panel B as a compressed configuration) is activated by mechanical force and "extension" of the central region (C). This would activate Src, induce tyrosine phosphorylation of the repetitive YxxP motifs, and recruit Crk through its SH2 domain. Further, by recruiting different proteins via the CrkSH3N, this signaling strategy would spatially integrate divergent signals, for example, after the recruitment of various GTPase pathways such as DOCK1, SOS, and C3G.

Figure 3

(A). Schematic structures of DOCK proteins. The SH3 domain, Docker Homology Region 1/CDM-Zizimin Homology1, Docker Homology Region 2/CDM Zizimin Homology 2, and Proline-rich sequences (shown in red) are indicated. The circular structure indicates the putative location of the Crk binding sites. Note that in DOCK5, Crk has been shown to bind to a non-canonical proline sequence. In DOCK2, Crk has been proposed to bind independent of proline. (B). Structure of the autoinhibited DOCK1/Crk complex. See text for details.

Figure 4

The multiple fates of Crk and Abl interactions. Crk binding to the proline-rich region of Abl induces Abl transactivation (indicated by circle enclosing The (P). Subsequent to Abl activation following Crk binding, Abl phosphorylates Crk on Tyr-221, causing dissociation of the Abl and Crk complex. The fate of activated Abl is not known, but some studies indicate that activated Abl is ubiquitinated and degraded by the proteosomal pathway.

Figure 5

SH3N and SH3C domain communication in chicken Crk II is mediated by interaction of CyPA. Prolyl cis-trans isomerization centered on P₂₃₈FY regulates intradomain communication between the SH3N and SH3C domains. In the cis configuration, the SH3C or Crk forms a closed structure over the SH3N, preventing its association with proline-containing binding partners. In the trans configuration, the linker and SH3N are extended, releasing negative regulation. Blue = SH3N; Orange = SH3C; Green = CypA.

Figure 6

Sequence alignment of various Crk species. Chicken Crk is unique in possessing a PFY motif. As alluded to in the text, this may imply that chicken Crk is uniquely regulated by prolyl cis-trans isomerization. Shown in the right side of the figure is a schematic representaion depicting accessibility in the different conformations.

Figure 7

NMR analysis of the structure of Crk II and pCrkII. (A) NMR structure of CrkII (1–304) and phosphorylated form of CrkII (1–228). SH2, SH3N, and SH3C are indicated as red, green, and blue colors. Inter SH3 core region (224–237) of CRKII is indicated as yellow. In pCrkII, interface of SH3N is covered with the sequence between SH2 and SH3N (122-133 as magenta). Phosphorylated residue of pYAQP is indicated as light blue. (B). Schematic structure of CrkII with SH3N target C3G. In the cytoplasm, equilibrium of CrkII alone and CrkII/C3G complex may be established.

Figure 8

Effect of Crk siRNA on morphology and biological activity of human ovarian cancer cell line MCAS. (A). Membrane ruffling is observed in the control MCAS cells (left panel), and siRNA for Crk suppressed these phenotypes (middle). When Crk is overexpressed, clear ruffling appeared (right, yellow color indicates Crk expression). (B). Control MCAS cells form metastatic nodules in the peritoneal cavity mimicking peritonitis carcinomatosa of human ovarian cancer patients when cells are injected into the peritoneal cavity of nude mice. H&E stain for lymphatic invasion of the peritoneal wall is displayed (left). Crk depleted cells by siRNA lose their malignant potential (right). [See additional file 2] (i) Human synovial sarcoma cell line Fuji with control siRNA was stimulated with HGF. (ii) Human synovial sarcoma cell line Fuji with CRK siRNA was stimulated with HGF. Note that the cells exhibit flat morphology and their movement is static.