NEK1 kinase domain structure and its dynamic protein interactome after exposure to Cisplatin

Abstract

NEK family kinases are serine/threonine kinases that have been functionally implicated in the regulation of the disjunction of the centrosome, the assembly of the mitotic spindle, the function of the primary cilium and the DNA damage response. NEK1 shows pleiotropic functions and has been found to be mutated in cancer cells, ciliopathies such as the polycystic kidney disease, as well as in the genetic diseases short-rib thoracic dysplasia, Mohr-syndrome and amyotrophic lateral sclerosis. NEK1 is essential for the ionizing radiation DNA damage response and priming of the ATR kinase and of Rad54 through phosphorylation. Here we report on the structure of the kinase domain of human NEK1 in its apo- and ATP-mimetic inhibitor bound forms. The inhibitor bound structure may allow the design of NEK specific chemo-sensitizing agents to act in conjunction with chemo- or radiation therapy of cancer cells. Furthermore, we characterized the dynamic protein interactome of NEK1 after DNA damage challenge with cisplatin. Our data suggest that NEK1 and its interaction partners trigger the DNA damage pathways responsible for correcting DNA crosslinks.

Comprehensive substrate specificity profiling of the human Nek kinome reveals unexpected signaling outputs

“…We next investigated whether introduction of an HRD+2 threonine and/or APE-4 isoleucine in a serine/threonine kinase would be sufficient to induce tyrosine-directed activity. These mutational experiments were conducted using Nek1, since its structure is known (Melo-Hanchuk et al, 2017), wild-type Nek1 did not efficiently phosphorylate peptide substrates on tyrosine in our experiments (Figure 3—figure supplement 2A), and, like all other Nek kinases except Nek10, Nek1 has an HRD+2 lysine and APE-4 proline (Figure 3E,H). However, murine Nek1 has been reported to be a dual-specificity kinase (Letwin et al, 1992), suggesting that Nek1 might have intrinsic tyrosine-phosphorylation capacity in specific contexts, and might therefore be more susceptible than other serine/threonine kinases to mutations that broaden phospho-acceptor specificity.…”

“…To rationalize the unusual selectivity for acidic residues displayed by Group 4 Nek kinases, we compared the X-ray crystal structures of the Nek1 and Nek7 kinase domains (Haq et al, 2015; Melo-Hanchuk et al, 2017). This revealed the presence of two basic regions in Nek7 that are absent from Nek1, and might accommodate acidic residues N-terminal of the phospho-acceptor site (Figure 6F).…”

Checking NEKs: Overcoming a Bottleneck in Human Diseases

“…Additionally, NEK1 is involved in cell cycle progression and many DNA Damage Response (DDR) pathways. NEK1 was shown to interact with several DNA repair proteins, suggesting that it plays a key role in the DNA damage response (DDR) [30]. Finally, it has been suggested that NEK1 is involved in gametogenesis, due to its high expression levels in meiotic tissues [17] and important roles in spermatogenesis [22] and spindle assembly in meiosis I [17].…”

Mitotic Regulation by NEK Kinase Networks

“…The protein used had a T162A mutation, the principal site of activating phosphorylation, and was therefore in an inactive state. Unlike Nek2, the activation loop of inactive Nek1 was fully ordered, albeit in a conformation that is incompatible with substrate binding (Melo-Hanchuk et al, 2017 ). The region of the activation loop in the vicinity of residue 162 formed an α-helix.…”