# ALPK3 in cardiac function and disease

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2022 · $395,000

## Abstract

PROJECT SUMMARY
Alpha protein kinase 3 (ALPK3, also known as MAK, MIDORI) is a novel atypical protein kinase highly
expressed in cardiac muscle. Biallelic truncating mutations in ALPK3 cause severe pediatric cardiomyopathy.
Deficiency of ALPK3 in gene-trap mutant mice has been reported to cause cardiomyopathy. However, little is
known as to the specific role of ALPK3 in cardiomyocytes, or molecular mechanisms by which loss of ALPK3
results in cardiomyopathy. Furthermore, it is yet to be determined whether ALPK3 is a true protein kinase or
functions as a pseudokinase. To address the cardiac role of ALPK3, we have generated a floxed ALPK3
mouse line and used it to generate ALPK3 global knockout (gKO), as well as constitutive (cKO) and inducible
(icKO) cardiac-specific knockout mouse models. Our preliminary data revealed that, similar to the published
ALPK3 gene-trap mutant, ALPK3 gKO and cKO mice develop early onset cardiomyopathy. However, unlike
the reported ALPK3 gene-trap mice, our ALPK3 gKO and cKO mice exhibited a more severe dilated
cardiomyopathy (DCM) leading to premature lethality. We also observed that adult ALPK3 icKO mice develop
DCM and heart failure. These observations strongly suggest that ALPK3 plays a critical role in both developing
and adult cardiomyocytes. To test the kinase activity of ALPK3, we performed in vitro kinase assays using
recombinant ALPK3 kinase domain. Surprisingly, we did not detect kinase activity. Moreover, we generated a
novel ALPK3 knock-in mouse model in which the catalytic lysine (invariant lysine 1420) essential for phospho-
transfer activity was mutated to arginine (ALPK3KR/KR), thereby disrupting putative ALPK3 kinase activity.
ALPK3KR/KR mice did not display any cardiac abnormalities. Together, these observations indicate that the
putative phospho-transfer activity of ALPK3 is not required for cardiac function. To study the role of the putative
kinase domain, we generated a mutant mouse model in which the two Zn2+-coordinating cysteine residues
critical for α-kinase domain structure were mutated to alanine residues (ALPK3CA/CA). ALPK3CA/CA mice
displayed DCM, albeit less severe and with delayed onset relative to the DCM observed in ALPK3 cKO mice.
The cardiac phenotype of ALPK3CA/CA mutants indicates that a structurally intact putative kinase domain in
ALPK3 is critical for cardiac function. Taken together, the foregoing observations lead us to the hypothesis that
ALPK3 plays an essential role in regulating cardiac function, and that although devoid of catalytic activity, the
putative kinase domain, and/or other domains of ALPK3, mediate protein interactions critical for cardiac
function. Our Specific Aims are: 1. Elucidate the role of ALPK3 in developing and adult myocardium and 2.
Decipher mechanisms underlying the requirement for the putative ALPK3 kinase domain, devoid of kinase
activity, in cardiac function.

## Key facts

- **NIH application ID:** 10360581
- **Project number:** 5R01HL146759-04
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Ju Chen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $395,000
- **Award type:** 5
- **Project period:** 2019-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10360581

## Citation

> US National Institutes of Health, RePORTER application 10360581, ALPK3 in cardiac function and disease (5R01HL146759-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10360581. Licensed CC0.

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