NEK7 Protein, Human

NEK7 (NIMA-related kinase 7) is a serine/threonine kinase belonging to the NEK family. It plays a crucial and specific role in regulating cell cycle progression, particularly in mitotic entry and spindle assembly. Structurally distinct from other NEK kinases, NEK7 contains an N-terminal catalytic kinase domain and a C-terminal regulatory domain. Its activity is tightly controlled by cell cycle-dependent phosphorylation and its interaction with NEK9. The NEK9/NEK6/NEK7 cascade is a key pathway for mitotic centrosome separation and spindle formation.

A landmark function of NEK7 is its essential role in activating the NLRP3 inflammasome, a critical component of the innate immune system. NEK7 directly interacts with NLRP3 downstream of various danger signals (e.g., ATP, crystalline substances, and pathogens). This interaction is required for NLRP3 inflammasome assembly and the subsequent activation of caspase-1, leading to the maturation and secretion of pro-inflammatory cytokines IL-1β and IL-18 and triggering pyroptosis, a form of inflammatory cell death. This discovery bridges cell cycle regulation with innate immunity. Dysregulation of NEK7 is implicated in auto-inflammatory diseases (e.g., cryopyrin-associated periodic syndromes) and gout, and is a potential target for treating NLRP3-related disorders. In cancer, aberrant NEK7 expression is linked to genomic instability and tumorigenesis due to its role in mitosis.

Assay protocol

Principle: The NEK7 assay is performed using the ADP-GloTM Kinase Assay kit which quantifies the amount of ADP produced by the NEK7 reaction. The ADP-GloTM Reagent is added to terminate the kinase reaction and to deplete the remaining ATP, and then the Kinase Detection Reagent is added to convert ADP to ATP and to measure the newly synthesized ATP using luciferase/luciferin reaction.

Materials

1.Kinase assay buffer(5X): 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2 and 0.5 mg/mL BSA, 250 μM DTT

2.Kinase assay buffer(1X): 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/mL BSA, 50 μM DTT

3.NEK7 Protein, Human

4.ADP-Glo Kinase Assay (Promega, Catalog # V6930)

5.Substrate: Casein substrate (Sinobiological, Catalog # C03-54N)

6.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

7.Plate Reader (PerkinElmer)

Produce

1.Prepare a substrate/ATP mixture as follows (25 μM example).

2. Dilute the NEK7 to 15 µg/mL, 7.5 µg/mL and 3.75 µg/mL in Kinase Assay Buffer (1x) and dispense 3 μL into each well of a 384-well plate.

3. Initiate the reaction by adding 2 μL of the detection system prepared in Step 1 to each well. Include a detection system with 3 μL Kinase Assay Buffer (1x) as Blank. The reaction volume is 5 μL.

4. Incubate the reaction at room temperature (22–25℃) for 40 minutes.

5.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25℃).

6.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25℃).

7.Read at luminescence, respectively in endpoint mode.

8.Calculate specific activity.

• Standard Curve

1.Dilute the ATP and ADP to 25 μM in Kinase Assay Buffer (1x).

2.Mix 25 μΜ ATP and 25 μM ADP to form an ATP+ADP solution provided below and dispense 5 μL into each well of a 384-well plate.

3.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25℃).

4.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25℃).

5.Read at luminescence, respectively in endpoint mode.

6.Detect optical signals and establish conversion curves.