1H NMR conformational study of sulfated and non-sulfated cholecystokinin fragment CCK27–33: Influence of the sulfate group on the peptide folding

¹H NMR study of cholecystokinin fragment (CCK_27–33) in (C²H₃)₂SO and in ²H₂O at different pH shows that sulfated (CCK₇) and non sulfated (NS-CCK₇) peptides are under preferentially folded conformations characterized by a β-turn including the sequence Gly-Trp-Met-Asp with a H-bond between the CO of Gly and the NH of Asp. This structure is probably stabilized by an ionic interaction between Tyr and Asp. Moreover, the N-terminal part of CCK₇ forms a C₇ structure with a weak H-bond between the CO of Gly and the NH of Trp. In this model all CCK₇ hydrophobic side chains are in close vicinity, far from the hydrophilic sulfate group. Full interaction with brain CCK₈ receptors could require both the sulfate group and the maintening of conformational constraints.     

Stress-activated protein kinases are negatively regulated by cell density.

Stimulation by UV irradiation, TNFalpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by confluency will restrict c-Jun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.