S100 proteins are EF hand type Ca2+ binding proteins thought to function in stimulus-response coupling by binding to and thereby regulating cellular targets in a Ca2+-dependent manner. To isolate such target(s) of the S100P protein we devised an affinity chromatography approach that selects for S100 protein ligands requiring the biologically active S100 dimer for interaction. Hereby we identify ezrin, a membrane/F-actin cross-linking protein, as a dimer-specific S100P ligand. S100P-ezrin complex formation is Ca2+ dependent and most likely occurs within cells because both proteins colocalize at the plasma membrane after growth factor or Ca2+ ionophore stimulation. The S100P binding site is located in the N-terminal domain of ezrin and is accessible for interaction in dormant ezrin, in which binding sites for F-actin and transmembrane proteins are masked through an association between the N- and C-terminal domains. Interestingly, S100P binding unmasks the F-actin binding site, thereby at least partially activating the ezrin molecule. This identifies S100P as a novel activator of ezrin and indicates that activation of ezrin's cross-linking function can occur directly in response to Ca2+ transients.
Ca2؉ -binding proteins of the S100 family participate in intracellular Ca 2؉ signaling by binding to and regulating specific cellular targets in their Ca 2؉ -loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca 2؉ -dependent and characterized by a dissociation constant of 0.2 M. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca 2؉
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