Contribution of Crk adaptor proteins to host cell and bacteria interactions

Abstract

The Crk adaptor family of proteins comprises the alternatively spliced CrkI and CrkII isoforms, as well as the paralog Crk-like (CrkL) protein, which is encoded by a different gene. Initially thought to be involved in signaling during apoptosis and cell adhesion, this ubiquitously expressed family of proteins is now known to play essential roles in integrating signals from a wide range of stimuli. In this review, we describe the structure and function of the different Crk proteins. We then focus on the emerging roles of Crk adaptors during Enterobacteriaceae pathogenesis, with special emphasis on the important human pathogens Salmonella, Shigella, Yersinia, and enteropathogenic Escherichia coli. Throughout, we remark on opportunities for future research into this intriguing family of proteins.

Figure 1

Schematic representation of Crk adaptor protein structure.

Figure 2

Crk alignments based on NCBI sequences by using the Clustal Omega program from the European bioinformatics institute (EMBL-EBI) (http://www.ebi.ac.uk/Tools/msa/clustalo/). (a) SH2 domains. Note the PRR in CrkII. (b) nSH3 domains. (c) Linker region containing the regulatory tyrosines (indicated by an asterisk). (d) cSH3 domains. Note the low homology of mouse CrkII.

Figure 3

Roles of Crk proteins in bacterial pathogenesis. (a) Salmonella enterica serovar Typhimurium manipulates Abl kinase phosphorylation of Crk adaptors to gain entry into the host cell. S. Typhimurium activates Abl kinase via T3SS effectors. Abl phosphorylates CrkII regulatory tyrosines. FAK and p130Cas are recruited to the FA-like complex where p130Cas interacts with CrkII, leading to cytoskeletal rearrangement and uptake of the bacterium. Abl activation by Salmonella effectors may also result in WAVE2 complex activation, in part through phosphorylation of Abi1. The WAVE2 complex is necessary for activation of Arp2/3 to induce actin polymerization at the site of lamellipodia formation. WAVE2 activation also requires interaction with Rho-GTPases (e.g., Rac). Salmonella effectors may also directly activate Rho-GTPases. It is unclear whether crosstalk between the two pathways exists (adapted from [19]). (b) Shigella T3SS effectors activate Abl/Arg to phosphorylate Crk adaptors. Abl/Arg phosphorylates and activates CrkII, which regulates the activity of Rho-GTPases (e.g., Rac and Cdc42). Cortactin is phosphorylated by Src and phosphocortactin interacts with CrkII. Cortactin can contribute to activation of N-WASP and the Arp2/3 complex, resulting in actin polymerization and membrane ruffling. Rho-GTPases can also activate Arp2/3 and N-WASP. On the contrary, Unc119 blocks Abl/Arg phosphorylation of Crk. (c) Yersinia uptake is mediated by β1-integrin signaling to Crk adaptors. The p130Cas-Crk interaction is promoted during Yersinia invasion. FAK and Src are also recruited to this complex. The p130Cas-Crk complex formation is coupled to Rho-GTPase (e.g., Rac) activation, leading to activation of N-WASP, actin polymerization, and bacterial entrance. Alternatively, Yersinia YadA can bind to host cell integrin indirectly through ECM proteins (e.g., fibronectin). This activates a signaling cascade involving FAK, p130Cas and Rho-GTPases, which ultimately lead to actin remodeling and Yersinia uptake. At low ECM concentrations, the invasin-integrin model predominates. At high ECM concentrations, the YadA-ECM-integrin model predominates. Yersinia likely uses a combination of invasin and YadA binding to gain entry into the cell. (d) Crk adaptor proteins are targeted by several EPEC effectors during infection. Tir is inserted into the host cell membrane where binds intimin on the bacterial surface, resulting in intimate attachment of E. coli to the host cell. Abl/Arg and Src family kinases (SFKs) phosphorylate Tir (“Tir insertion”), resulting in the recruitment of Nck. Nck recruits and activates N-WASP leading to Arp2/3 complex-mediated actin polymerization and pedestal formation. Abl/Arg phosphorylates CrII/CrkL (“initiation of pedestal formation”; see [20] for details). Tir levels are reduced in the absence of Nck (dashed circle; Nieto-Pelegrin et al, in press). Other FA components localize to pedestals (e.g., FAK, p130Cas, vinculin, and paxillin; “Actin polymerization/Pedestal formation”), though some are degraded by the EspC effector (dashed lines; [21]). During infection, the transcriptional regulator, NF-κB, becomes activated and translocates to the nucleus. In addition, RPS3 is phosphorylated by IKKβ and translocates to the nucleus via importin-α, where RPS3 acts as a “specifier” for NF-κB to select for and regulate a specific subset of innate immune response genes. The effector NleH1 interacts with CrkL and subsequently prevents the phosphorylation of RPS3, thus blocking RPS3 nuclear translocation and inhibiting NF-κB activation and the innate immune response. In the absence of CrkL (dashed lines), NleH1 cannot block RPS3 translocation to the nucleus. Model partially adapted from [22]. Other translocated effectors include mitochondrial associated protein (Map) and EPEC-secreted proteins (Esp) H, F, G, and Z [23] that are encoded within a pathogenicity island termed the locus of enterocyte effacement (LEE) [24].