Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin.

Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.

Monitoring the epidemic of West Nile virus in equids in Spain, 2020-2021.

The largest epidemic of West Nile virus (WNV) reported ever in Spain in both humans and equines occurred in 2020, affecting 77 humans and 139 equine herds. Here, we aimed to monitor the outbreaks detected in equid herds in Andalusia (southern Spain), the Spanish region where 89.9% of the outbreaks were reported, and to evaluate the virus circulation and risk factor associated with WNV exposure in the affected herds. The first WNV case was detected in mid-July 2020, the number of outbreaks peaked in mid-August and the last one was confirmed on 26th October 2020. WNV lineage 1 was detected in 12 clinically affected horses using real time RT-PCR. Molecular analysis evidenced high nucleotide identity with WNV sequences obtained from humans, birds and mosquitoes from Spain and Italy between 2020 and 2022. Between five and eight months after the WNV epidemic, a total of 724 equids (including 485 unvaccinated and 239 vaccinated animals) from 113 of the 125 affected herds in Andalusia were sampled. IgM and IgG antibodies against WNV were detected in 1.6% (8/485; 95%IC: 0.0-2.5) and 61.9% (300/485; 95%IC: 58.3-65.5) of the unvaccinated individuals, respectively. The seropositivity in vaccinated horses was 86.6% (207/239). The main risk factors associated with WNV exposure in unvaccinated equids were the breed (crossbreed), the location of animals in spring-summer (outside), and the presence of natural water ponds close to the surveyed herds. The high individual seroprevalence obtained in the affected herds indicates that WNV circulation was more widespread than the reported by passive surveillance during the WNV epidemic in 2020. The re-emergence of WNV in 2020 in southern Spain evidenced the needed to improve integrated surveillance systems, minimizing the impact of future cases in equids and humans in high-risk areas.

Nitric oxide-induced epidermal growth factor-dependent phosphorylations in A431 tumour cells.

Nitric oxide (NO*) strongly inhibits the proliferation of human A431 tumour cells. It also inhibits tyrosine phosphorylation of a 170-kDa band corresponding to the epidermal growth factor receptor (EGFR) and induces the phosphorylation at tyrosine residue(s) of a 58-kDa protein which we have denoted NOIPP-58 (nitric oxide-induced 58-kDa phosphoprotein). The NO*-induced phosphorylation of NOIPP-58 is strictly dependent on the presence of EGF. Phosphorylation of NOIPP-58 and inhibition of the phosphorylation of the band corresponding to EGFR are both cGMP-independent processes. We also demonstrate that the p38 mitogen-activated protein kinase (p38MAPK) pathway is activated by NO* in the absence and presence of EGF, whereas the activity of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and the c-Jun N-terminal kinase 1/2 (JNK1/2) pathways are not significantly affected or are slightly decreased, respectively, on addition of this agent. Moreover, we show that the p38MAPK inhibitor, SB202190, induces rapid vanadate/peroxovanadate-sensitive dephosphorylation of prephosphorylated EGFR and NOIPP-58. We propose that the dephosphorylation of both NOIPP-58 and EGFR are mediated by a p38MAPK-controlled phosphotyrosine-protein phosphatase (PYPP). Activation of the p38MAPK pathway during nitrosative stress probably prevents the operation of this PYPP, allowing NOIPP-58, and in part EGFR, to remain phosphorylated and therefore capable of generating signalling events.