Potential involvement of S147P and P21H SNP sites in nmMLCK1 in phosphorylation-dependent binding of 14-3-3 proteins.

<p>(A) Sequence alignment of human nmMLCK1 (wild type and SNPs) with the consensus of 14-3-3 binding modes and selected protein kinase substrates as well as its murine ortholog. The “S” or “T” in <i>red</i> indicates a phophorylation site, with underline indicating a predicted site (<i>black</i> underlined) or a confirmed site (<i>red</i> underlined). The “R” or “K” in <i>blue</i> and the “P” in <i>green</i> indicate their potential involvement in key binding recognition. The “<u>P</u>” indicates a “P” that may be missing and hence nonessential. The “X” in <i>black</i> indicates any amino acid. The “<u>X</u>” indicates an “X” that may be missing and hence nonessential. The consensus of GSK3 substrate (<u>S</u>XXX<u>S</u>, the 2nd <u>S</u> representing a pre-existing pSer or pThr) is shown in repeat in order to align with multiple potential phosphorylation sites in nmMLCK1. While not shown, additional multiple alignments of S147, S18, and other nearby Ser residues including S16, S26, S145 and S154, are possible with the highly variable consensus substrate R<u>XX</u>X<u>S</u> of the AGC group of protein kinases that include PKA, PKG and PKC families, suggesting a complex regulation of nmMLCK via these SNP-embracing loops by different kinase-mediated phosphorylation and subsequent binding to 14-3-3 proteins. (B) Molecular modeling of the 1-252aa segment of nmMLCK1 [template: deleted in colorectal cancer (DCC) (PDBID: 3lafA)] revealing localization of S147P and P21H SNP sites in separate loops at the two ends of the single, 1st IGc2 domain (with side chains of some loop residues of interest shown), despite that the modeling of loop conformations may be of poor quality. (C) Immunoprecipitation (IP) of Flag-tagged nmMLCK1 wild type using Flag-M5 anitibody followed by western blot using pan-14-3-3 antibody indicating the binding of 14-3-3 proteins to nmMLCK1 before and after S1P stimulation. Note: In the IP result shown, the bottom 14-3-3 bands correspond to 24 kDa and the top 14-3-3 bands correspond to 27 kDa.