Anti-NEK6 (aa 1-100) polyclonal antibody

The AGC family of protein kinases, which includes isoforms of protein kinase B (also known as Akt), ribosomal S6 protein kinase (SW), and serum- and glucocorticoid-induced protein kinase (SGK) are activated in response to many extracellular signals and play key roles in regulating diverse cellular processes. They are activated by the phosphorylation of the T loop of their kinase domain by the 3-phosphoinositide-dependent protein kinase-1 and by phosphorylation of a residue located C-terminal to the kinase domain in a region termed the hydrophobic motif. Recent work has implicated the NIMA (never in mitosis, gene A)-related kinase-6 (NEK6) as the enzyme that phosphorylates the hydrophobic motif of S6K1 in vivo. Here we demonstrate that in addition to phosphorylating S6K1 and SGK1 at their hydrophobic motif, NEK6 also phosphorylates S6K1 at two other sites and phosphorylates SGK1 at one other site in vitro. Employing the Jerini pepSTAR method in combination with kinetic analysis of phosphorylation of variant peptides, we establish the key substrate specificity determinants for NEK6. Our analysis indicates that NEK6 has a strong preference for Leu 3 residues N-terminal to the site of phosphorylation. Its mutation to either Ile or Val severely reduced the efficacy with which NEK6-phosphorylated peptide substrates, and moreover, mutation of the equivalent Leu residue in S6K1 or SGK1 prevented phosphorylation of their hydrophobic motifs by NEK6 in vitro. However, these mutants of S6K1 or SGK1 still became phosphorylated at their hydrophobic motif following insulin-like growth factor-1 stimulation of transfected 293 cells. This study provides the first description of the basis for the substrate specificity of NEK6 and indicates that NEK6 is unlikely to be responsible for the IGF1-induced phosphorylation of the hydrophobic motif of S6K, SGK, and protein kinase B isoforms in vivo.

Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1 Delta ARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1 Delta ARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression.