Characterization of hNek6 Interactome Reveals an Important Role for Its Short N-Terminal Domain and Colocalization with Proteins at the Centrosome

Abstract

Physical protein-protein interactions are fundamental to all biological processes and are organized in complex networks. One branch of the kinome network is the evolutionarily conserved NIMA-related serine/threonine kinases (Neks). Most of the 11 mammalian Neks studied so far are related to cell cycle regulation, and due to association with diverse human pathologies, Neks are promising chemotherapeutic targets. Human Nek6 was associated to carcinogenesis, but its interacting partners and signaling pathways remain elusive. Here we introduce hNek6 as a highly connected member in the human kinase interactome. In a more global context, we performed a broad data bank comparison based on degree distribution analysis and found that the human kinome is enriched in hubs. Our networks include a broad set of novel hNek6 interactors as identified by our yeast two-hybrid screens classified into 18 functional categories. All of the tested interactions were confirmed, and the majority of tested substrates were phosphorylated in vitro by hNek6. Notably, we found that hNek6 N-terminal is important to mediate the interactions with its partners. Some novel interactors also colocalized with hNek6 and γ-tubulin in human cells, pointing to a possible centrosomal interaction. The interacting proteins link hNek6 to novel pathways, for example, Notch signaling and actin cytoskeleton regulation, or give new insights on how hNek6 may regulate previously proposed pathways such as cell cycle regulation, DNA repair response, and NF-κB signaling. Our findings open new perspectives in the study of hNek6 role in cancer by analyzing its novel interactions in specific pathways in tumor cells, which may provide important implications for drug design and cancer therapy.

Aspergillus niger β-Glucosidase Has a Cellulase-like Tadpole Molecular Shape

“…The protein bands were removed from the gel and were submitted to in-gel trypsin (20 ng/l), chymotrypsin (100 ng/l), and pepsin A (75 ng/l) digestion as described previously (19).…”

Cell cycle regulation by the NEK family of protein kinases

“…NEK6 and NEK7 notably lack a C-terminal domain. They consist solely of a kinase domain with a short N-terminal extension that might be important for substrate recognition (Vaz Meirelles et al, 2010). Indeed, the non-catalytic regions of the other NEKs almost certainly contribute to substrate recognition, as well as regulation, and this is illustrated, for example, by the recognition of NEK6 and NEK7 by a sequence in the C-terminus of NEK9 (Roig et al, 2002).…”

Molecular Determinants of Hormone-Refractory Prostate Cancer

“…The NEK6 interactome as shown in Table 3 includes many genes putatively involved in immune signaling including IKBKB, DDX3X, ILF2, PRDX4, RHOC, S100A8, S100A9, and TOLLIP; however, none of these proteins act as direct NEK6 substrates in vitro in comparison to the potent substrates CK1α and YES1 (Supplemental Figure 6). In addition, NEK6 has previously been reported to interact with RELB (Vaz Meirelles et al, 2010) and increase the activity of an NFκB promoter (Matsuda et al, 2003), and it has also been previously reported to phosphorylate STAT3 (Jeon et al, 2010); however, RELA, RELB, STAT1, STAT3, and STAT5 are also not phosphorylated to an appreciable extent by NEK6 in our in vitro kinase assay. Thus, the impact of NEK6 on immune signaling may be indirect or the relevant substrate(s) have yet to be identified.…”

“…However, there is little evidence from our unbiased studies suggesting any of these pathways as the predominant mechanism of NEK6-mediated castration resistance in our model. Previous proteomic work (Vaz Meirelles et al, 2010) identifies multiple interacting partners and substrates that implicate NEK6 in a variety of signaling pathways including cell cycle, cytoskeleton organization, DNA repair, Notch and NFκB pathways. In our studies, the gene expression signature mediated by NEK6 overexpression in the context of castration implicates differentiation and immune processes in conferring castration resistance to tumors.…”

“…However, a canonical NEK6 motif (Vaz Meirelles et al, 2010) -5 ). This suggests that this peptide sequence is a true description of the NEK6 phosphorylation motif, and that the 8 proteins with phosphopeptides of this form detected here 8 (FOXJ2, HUWE1, NCOA5, KRT18, TRA2B, HNRNPM, HNRNPA2B1, ZNF326) are bona fide in vivo NEK6 substrates.…”