Dataset Information

AKAP6 and phospholamban colocalize and interact in HEK-293T cells and primary murine cardiomyocytes.

ABSTRACT: Phospholamban (PLN) is an important Ca2+ modulator at the sarcoplasmic reticulum (SR) of striated muscles. It physically interacts and inhibits sarcoplasmic reticulum Ca2+ ATPase (SERCA2) function, whereas a protein kinase A (PKA)-dependent phosphorylation at its serine 16 reverses the inhibition. The underlying mechanism of this post-translational modification, however, remains not fully understood. Using publicly available databases, we identified A-kinase anchoring protein 6 (AKAP6) as a candidate that might play some roles in PLN phosphorylation. Immunofluorescence showed colocalization between GFP-AKAP6 and PLN in transfected HEK-293T cells and cultured mouse neonatal cardiomyocytes (CMNCs). Co-immunoprecipitation confirmed the functional interaction between AKAP6 and PLN in HEK-293T and isolated adult rat cardiomyocytes in response to isoproterenol stimulation. Functionally, AKAP6 promoted Ca2+ uptake activity of SERCA1 in cotransfected HEK-293T cells despite the presence of PLN. These results were further confirmed in adult rat cardiomyocytes. Immunofluorescence showed colocalization of both proteins around the perinuclear region, while protein-protein interaction was corroborated by immunoprecipitation of the nucleus-enriched fraction of rat hearts. Our findings suggest AKAP6 as a novel interacting partner to PLN in HEK-293T and murine cardiomyocytes.

SUBMITTER: Hakem Zadeh F

PROVIDER: S-EPMC6642276 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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AKAP6 and phospholamban colocalize and interact in HEK-293T cells and primary murine cardiomyocytes.

Hakem Zadeh Farigol F Teng Allen C T ACT Kuzmanov Uros U Chambers Paige J PJ Tupling Allan R AR Gramolini Anthony O AO

Physiological reports 20190701 14

Phospholamban (PLN) is an important Ca<sup>2+</sup> modulator at the sarcoplasmic reticulum (SR) of striated muscles. It physically interacts and inhibits sarcoplasmic reticulum Ca<sup>2+</sup> ATPase (SERCA2) function, whereas a protein kinase A (PKA)-dependent phosphorylation at its serine 16 reverses the inhibition. The underlying mechanism of this post-translational modification, however, remains not fully understood. Using publicly available databases, we identified A-kinase anchoring prote ...[more]

PMID: 31325238

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Project description:UNLABELLED:We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. IMPORTANCE:Tick-borne flaviviruses are significant infectious agents that cause serious disease and death in humans worldwide. Infections are characterized by severe neurological symptoms, such as meningitis and encephalitis. A high percentage of people who get infected and recuperate from the acute phase of infection continue to suffer from chronic debilitating neurological sequelae, most likely as a result of nervous tissue damage, viral persistence, or both. However, little is known about mechanisms of viral persistence. Therefore, we undertook studies to investigate the persistence of Langat virus, a member of the tick-borne flavivirus group, in a mammalian cell line. Using next-generation sequencing, we determined that defective viral genomes do not play a role in the initiation of persistence, but their occurrence seems to be nonstochastic and could play a role in the maintenance of viral persistence via the expression of a novel envelope-NS1 fusion protein.

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Dataset Information

AKAP6 and phospholamban colocalize and interact in HEK-293T cells and primary murine cardiomyocytes.

Publications

AKAP6 and phospholamban colocalize and interact in HEK-293T cells and primary murine cardiomyocytes.

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