Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

Abstract

Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.

Characterisation of cellular effects of Burkholderia pseudomallei Cycle inhibiting factor (Cif)

“…We found that one additional cellular target of Cif is the ERK MAPK pathway. We have recently shown that activation of ERK by Cif is CRL independent ( Ng et al, 2017 ). However, ERK activation was not involved in the Cif-mediated stimulation of B. thailandensis replication.…”

Burkholderia pseudomallei BipD modulates host mitophagy to evade killing

“…B. pseudomallei TssM, the homologous protein of B. mallei TssM, acts as a DUB, and has been currently identified to inhibit the activation of NF-kB by promoting its substrates TRAF-3, TRAF-6 and IκBα deubiquitination 48 . In addition, CHBP (Cif homolog from B. pseudomallei ) can catalyze the deamidation of Gln 40 in ubiquitin and NEDD8 to inhibit Cullin RING E3 ubiquitin ligases (CRL) activity in vitro 49 , and further be verified to activate MAPK/ERK signaling in cellular assays which is independent of CRL inhibition 50 , 51 . Of note, many pathogenic B. pseudomallei strains lack the Cif gene, suggesting that CHBP is not an essential virulence factor in B. pseudomallei 51 .…”

Cycle-Inhibiting Factor Is Associated with Burkholderia pseudomallei Invasion in Human Neuronal Cells

“…Protein ubiquitination also plays crucial roles in controlling eukaryotic cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases [ 51 ]. B. pseudomallei Cif has been shown to modify host central signaling pathways by activating MAPK/ERK signaling to induce the phosphorylation of the pro-apoptotic protein Bim (Bcl-2 interacting mediator of cell death), and potentially leading to a proapototic effect [ 34 ]. Additionally, Cif was shown to increase basal NF-κB activity and stimulate the intracellular replication of the B. thailandensis [ 35 ].…”

“…The modus operandi of Cif is known to deamidate Nedd8, an ubiquitin-like protein, which causes the inhibition of Cullin E3 ubiquitin ligases (CRL), and consequently induces cell cycle arrest [ 31 , 32 , 33 ]. B. pseudomallei Cif can function as a potent activator of MAPK/ERK signaling and has CRL independent effects to counter the pro-apoptotic effects [ 34 ]. However, whether Cif employs additional roles on host cells that are crucial for bacterial pathogenesis is currently poorly understood.…”