Crystal structure analysis and solution studies of human Lck-SH3; zinc-induced homodimerization competes with the binding of proline-rich motifs.

Lehrstuhl für Biotechnik, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
Journal of Molecular Biology (Impact Factor: 3.96). 03/2007; 365(5):1417-28. DOI: 10.1016/j.jmb.2006.10.058
Source: PubMed

ABSTRACT In cytosolic Src-type tyrosine kinases the Src-type homology 3 (SH3) domain binds to an internal proline-rich motif and the presence or the absence of this interaction modulates the kinase enzymatic activity. The Src-type kinase Lck plays an important role during T-cell activation and development, since it phosphorylates the T-cell antigen receptor in an early step of the activation pathway. We have determined the crystal structure of the SH3 domain from Lck kinase at a near-atomic resolution of 1.0 A. Unexpectedly, the Lck-SH3 domain forms a symmetrical homodimer in the crystal and the dimer comprises two identical zinc-binding sites in the interface. The atomic interactions formed across the dimer interface resemble strikingly those observed between SH3 domains and their canonical proline-rich ligands, since almost identical residues participate in both contacts. Ultracentrifugation experiments confirm that in the presence of zinc ions, the Lck-SH3 domain also forms dimers in solution. The Zn(2+) dissociation constant from the Lck-SH3 dimer is estimated to be lower than 100 nM. Moreover, upon addition of a proline-rich peptide with a sequence corresponding to the recognition segment of the herpesviral regulatory protein Tip, competition between zinc-induced homodimerization and binding of the peptide can be detected by both fluorescence spectroscopy and analytical ultracentrifugation. These results suggest that in vivo, too, competition between Lck-SH3 homodimerization and binding of regulatory proline-rich sequence motifs possibly represents a novel mechanism by which kinase activity is modulated. Because the residues that form the zinc-binding site are highly conserved among Lck orthologues but not in other Src-type kinases, the mechanism might be peculiar to Lck and to its role in the initial steps of T-cell activation.

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Die Untersuchungen zeigen, dass TCR-stimulierte T-Zellen intrazellulär Zink freisetzen, welches selbst keinen Einfluss auf die Signalweiterleitung in die Zelle hat. Werden die Zellen allerdings mit Zink/Pyrithion stimuliert, werden die Kinasen Lck und p38 aktiviert, während die TCR-stimulierte ERK1/2-Phosphorylierung inhibiert wird. Um diesen differentiellen Zink-Effekt aufzuklären, wurden im Anschluss verschiedene, in anderen Zelltypen bereits bekannte Wirkmechanismen von Zink hinsichtlich ihrer Gültigkeit in T-Zellen untersucht. Die Lck wird durch Zink in ihrer Aktivität auf unterschiedlichen Ebenen beeinflusst. In dieser Arbeit konnte gezeigt werden, dass die Aktivierung nach Zink/Pyrithion Stimulation zusätzlich abhängig ist von der Proteintyrosinphosphatase CD45. Des Weiteren ist der beobachtete Zink-Effekt unabhängig von CD3 und stellt damit ein intrazelluläres Ereignis dar. Eine Inhibition von MAPK Phosphatasen durch Zink als mögliche Erklärung für die p38-Aktivierung ist auszuschließen. Eine Zink-induzierte Phosphorylierung der p38 durch die alternative Aktivierung konnte durch Inhibitorstudien und den Einsatz von defizienten Zelllinien ebenfalls ausgeschlossen werden. Auch die Beteiligung des PKA-Signalweges konnte unter Verwendung verschiedener Aktivatoren und Inhibitoren ausgeschlossen werden. Nach Überprüfung unterschiedlicher Signalwege muss festgehalten werden, dass der molekulare Mechanismus des beobachteten Zink-Effektes in T-Zellen von den gängigen Mechanismen in anderen Zellen abweicht. Trotz unvollständiger Aufklärung des Mechanismus konnte ein Zusammenhang zwischen der Induktion von Th1-Antworten und der Aktivierung der p38/CREB Kaskade nachgewiesen werden. Durch die vorliegende Arbeit wird erstmals der Einfluss von Zink auf Th1-Zellen mit der Aktivierung der MAPK p38 in Zusammenhang gebracht. Die weitere Aufklärung des molekularen Mechanismus könnte Einfluss auf die Behandlung T-Zell vermittelter Erkrankungen haben und demnach neue Therapieperspektiven eröffnen. Zinc is an essential trace element with a variety of cellular functions in all organ systems. Disturbance of the cellular zinc availability by zinc deficiency leads to multiple disorders. Predominantly the cell-mediated immune response is influenced by zinc deprivation, and here in particular T-cell mediated functions. For example, the Th1/Th2 balance is disturbed leading to a reduced production of Th1 cytokines such as IFN-gamma and IL-2. Zinc supplementation in vivo causes reconstitution of Th1-mediated responses. To achieve a broad picture about the role of zinc in T-cell signal transduction, phosphorylation of several effector kinases in primary human T-cells and in the human T-cell line Jurkat was investigated. CD3-mediated activation and stimulation by zinc and the ionophore pyrithione led to differential activation of kinase Lck, MAPK p38, JNK1/2 and ERK1/2. Experiments showed that CD3-stimulated T-cells release intracellular zinc which has no effect on signal transduction itself. However, if cells were stimulated with zinc and pyrithione, the kinases Lck and p38 were activated, while CD3-induced ERK1/2 phosphorylation was reduced. To elucidate this differential zinc effect, several known zinc-mediated signalling mechanisms were investigated for their validity in T-cells. Lck is influenced by zinc in its activity at different levels. Experiments with Lck-negative cells showed that activation after stimulation with zinc and pyrithione additionally depends on protein tyrosine phosphatase CD45. Furthermore the observed zinc effect is independent from CD3 signalling and represents an intracellular event. MAPK phosphatase inhibition by zinc could be excluded as one possible explanation for p38 activation. Zinc induced phosphorylation of p38 by alternative activation mediated by Lck and ZAP70 could be also eliminated by inhibitor studies and the use of Lck and ZAP70 deficient cell lines. Further on, the involvement of the PKA signalling pathway was no explanation for the observed differential zinc effect, confirmed by the use of different inhibitors and activators. After verification of several signalling pathways, the molecular pathway of the observed zinc effect in T-cells seems to differ from established pathways in other cells. Although the mechanism could not be elucidated, it could be demonstrated that there is a connection between the induction of Th1 answers and the activation of p38/CREB cascade. For the first time the impact of zinc on Th1 cells could be linked to p38 activation. 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