The C-terminal sequence of MEK2 binds to the PDZ repeats of hDlg. Peptides corresponding to the C-terminal end of human MEK2 (2CT) or human MEK1 (1CT), and a randomized version of 2CT (2RD) were assayed for binding to a polypeptide comprising PDZ1-PDZ2 of hDlg fused to GST (PDZ1-2). Bound fractions were analyzed by SELDI on H4 spots on a ProteinChip array (see material and methods). The area under the peak (arbitrary units) was proportional to the amount of bound peptide. (A) While 2CT bound to GST-PDZ1-2, no detectable peptide peak was observed when either 1CT or 2RD were tested for binding to GST-PDZ1-2; an assay with an unrelated GST fusion protein (α14) established the binding specificity of 2CT. (B) 2CT also bound to individual PDZ repeats fused with GST (PDZ1, left and PDZ2, middle). In this assay, the analysis of 2CT directly spotted on the ProteinChip array showed that protein-bound 2CT and free 2CT occupied the same position on MALDI spectra (right). Preliminary experiments showed that both 1CT and 2RD can be detected by SELDI (data not shown).
Gaudet et al. BMC Cell Biology 2011 12:55 doi:10.1186/1471-2121-12-55