Essential role of obscurin kinase-1 in cardiomyocyte coupling via N-cadherin phosphorylation.

Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains 2 enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc, the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin colocalize at cell junctions in embryonic rat ventricular myocytes (ERVMs), and their codistribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain, with an apparent Kcat of approximately 5.05 min-1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVMs markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.

DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex.

The Rad9-Rad1-Hus1 checkpoint clamp activates the DNA damage response and promotes DNA repair. DNA loading on the central channel of the Rad9-Rad1-Hus1 complex is required to execute its biological functions. Because Rad9A has the highest DNA affinity among the three subunits, we determined the domains and functional residues of human Rad9A that are critical for DNA interaction. The N-terminal globular domain (residues 1-133) had 3.7-fold better DNA binding affinity than the C-terminal globular domain (residues 134-266) of Rad9A1-266. Rad9A1-266 binds DNA 16-, 60-, and 30-fold better than Rad9A1-133, Rad9A134-266, and Rad9A94-266, respectively, indicating that different regions cooperatively contribute to DNA binding. We show that basic residues including K11, K15, R22, K78, K220, and R223 are important for DNA binding. The reductions on DNA binding of Ala substituted mutants of these basic residues show synergistic effect and are dependent on their residential Rad9A deletion constructs. Interestingly, deletion of a loop (residues 160-163) of Rad9A94-266 weakens DNA binding activity by 4.1-fold as compared to wild-type (WT) Rad9A94-266. Cellular sensitivity to genotoxin of rad9A knockout cells is restored by expressing WT-Rad9Afull. However, rad9A knockout cells expressing Rad9A mutants defective in DNA binding are more sensitive to H2O2 as compared to cells expressing WT-Rad9Afull. Only the rad9A knockout cells expressing loop-deleted Rad9A mutant are more sensitive to hydroxyurea than cells expressing WT-Rad9A. In addition, Rad9A-DNA interaction is required for DNA damage signaling activation. Our results indicate that DNA association by Rad9A is critical for maintaining cell viability and checkpoint activation under stress.

Mammalian MutY Homolog (MYH or MUTYH) is Critical for Telomere Integrity under Oxidative Stress.

Telomeres consist of special features and proteins to protect the ends of each chromosome from deterioration and fusion. The telomeric DNA repeats are highly susceptible to oxidative damage that can accelerate telomere shortening and affect telomere integrity. Several DNA repair factors including MYH/MUTYH DNA glycosylase, its interacting partners Rad9/Rad1/Hus1 checkpoint clamp, and SIRT6 aging regulator, are associated with the telomeres. MYH prevents C:G to A:T mutation by removing adenine mispaired with a frequent oxidative DNA lesion, 8-oxoguanine. Here, we show that hMYH knockout (KO) human HEK-293T cells are more sensitive to H2O2 treatment, have higher levels of DNA strand breaks and shorter telomeres than the control hMYH +/+ cells. SIRT6 foci increase at both the global genome and at telomeric regions in H2O2-treated hMYH +/+ cells. However, in untreated hMYH KO HEK-293T cells, SIRT6 foci only increase at the global genome, but not at the telomeric regions. In addition, the hMYH KO HEK-293T cells have increased extra-chromosomal and intra-chromosomal telomeres compared to the control cells, even in the absence of H2O2 treatment. After H2O2 treatment, the frequency of extra-chromosomal telomeres increased in control HEK-293T cells. Remarkably, in H2O2-treated hMYH KO cells, the frequencies of extra-chromosomal telomeres, intra-chromosomal telomeres, and telomere fusions are further increased. We further found that the sensitivity to H2O2 and shortened telomeres of hMYH KO cells, are restored by expressing wild-type hMYH, and partially rescued by expressing hMYHQ324H mutant (defective in Hus1 interaction only), but not by expressing hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions). Thus, MYH interactions with SIRT6 and Hus1 are critical for maintaining cell viability and telomeric stability. Therefore, the failure to coordinate 8-oxoG repair is detrimental to telomere integrity.

An ordered assembly of MYH glycosylase, SIRT6 protein deacetylase, and Rad9-Rad1-Hus1 checkpoint clamp at oxidatively damaged telomeres.

In the base excision repair pathway, MYH/MUTYH DNA glycosylase prevents mutations by removing adenine mispaired with 8-oxoG, a frequent oxidative lesion. MYH glycosylase activity is enhanced by Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp and SIRT6 histone/protein deacetylase. Here, we show that MYH, SIRT6, and 9-1-1 are recruited to confined oxidatively damaged regions on telomeres in mammalian cells. Using different knockout cells, we show that SIRT6 responds to damaged telomeres very early, and then recruits MYH and Hus1 following oxidative stress. However, the recruitment of Hus1 to damaged telomeres is partially dependent on SIRT6. The catalytic activities of SIRT6 are not important for SIRT6 response but are essential for MYH recruitment to damaged telomeres. Compared to wild-type MYH, the recruitment of hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions), but not hMYHQ324H mutant (defective in Hus1 interaction only), to damaged telomeres is severely reduced. The formation of MYH/SIRT6/9-1-1 complex is of biological significance as interrupting their interactions can increase cell's sensitivity to H2O2 and/or elevate cellular 8-oxoG levels after H2O2 treatment. Our results establish that SIRT6 acts as an early sensor of BER enzymes and both SIRT6 and 9-1-1 serve critical roles in DNA repair to maintain telomere integrity.