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. 2023 Dec 5;148(23):1911-1913.
doi: 10.1161/CIRCULATIONAHA.123.065993. Epub 2023 Dec 4.

ALPK3 Functions as a Pseudokinase

Wei Feng #  1 , Julius Bogomolovas #  1 , Li Wang  1 , Mengchen Li  1 , Ju Chen  1
Affiliations

ALPK3 Functions as a Pseudokinase

Wei Feng et al. Circulation. .
No abstract available

Keywords: ALPK3; CRISPR/Cas9; cardiomyopathy; kinase activity; pseudokinase.

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Conflict of interest statement

Disclosures J.C. consults for LEXEO Therapeutics and Morphic Therapeutic. J.B. consults for Rocket Pharmaceuticals on image analysis. The other authors report no conflicts.

Figures

Figure 1.
Figure 1.. ALPK3 functions as a pseudokinase.
(A) 3D alignment of catalytic domains of ALPK3 (AlphaFold) and TRPM7 (PDB ID: 1IAH). Positions of identical ADP-interacting residues are in green, differing ones in red. Position of the invariant catalytic lysine residue is marked in the enlarged nucleotide binding pocket. (B) In vitro phosphorylation assay of murine TRPM7 and murine ALPK3 catalytic domains on heat inactivated H9c2 (left panel) and hiPSC-CM (right panel) cell lysates. Coomassie blue stain (top) and autoradiography of γ-32P (middle) showing phosphorylated cell lysate substrate are shown. White asterisks indicate the position of the added recombinant protein kinase bands on the gel. Presence of SQSTM1 in H9c2 and hiPSC-CM cell lysates is confirmed by western blotting (bottom). (C) Schematics of CRISPR-Cas9 strategy to generate ALPK3 K1420R knock-in mice. (D) Sanger sequencing confirming mutation of AAA (encoding lysine) to AGG (encoding arginine) in ALPK3 KR knock-in mice. (E) qRT-PCR assessment of ALPK3 mRNA levels in the hearts of +/+ (N = 4) and KR/KR (N = 4) mice using primers targeting exons 2–4, 10–12, and 12–14 was performed. Each dot represents the value of ALPK3 mRNA levels obtained with qRT-PCR, with the average value of ALPK3 levels in +/+ mouse hearts arbitrarily set as 100%. Mean ± standard error of the mean (SEM) is shown. NS, not significant by Student’s t-test. (F) Echocardiography assessment of cardiac chamber size and contractile function of +/+ (N = 3–5) and KR/KR (N = 4–8) mice. Left ventricle internal dimension at diastole (LVIDd) and left ventricle internal dimension at systole (LVIDs) are used for measurement of chamber size. Fraction shortening (FS) is used for measurement of contractile function. Mean ± standard error of the mean (SEM) was shown. NS, not significant by Student’s t-test. (G) Representative western blotting of phosphorylated (T269/S272) and total SQSTM1 in +/+ and KR/KR mice. GAPDH is shown for loading control. (H) Quantification of p-SQSTM1 (T269/S272) to total SQSTM1 ratio from western blotting of phosphorylated (T269/S272) and total SQSTM1 in +/+ and KR/KR mice (N = 9 for each group). Mean ± standard error of the mean (SEM) was shown. NS, not significant by Student’s t-test.
See this image and copyright information in PMC

References

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