Mira Sastri

The Rate of NF-κB Nuclear Translocation Is Regulated by PKA and A Kinase Interacting Protein 1

The mechanism of PKAc-dependent NF-κB activation and subsequent translocation into the nucleus is not well defined. Previously, we showed that A kinase interacting protein 1 (AKIP1) was important for binding and retaining PKAc in the nucleus. Since then, other groups have demonstrated that AKIP1 binds the p65 subunit of NF-κB and regulates its transcriptional activity through the phosphorylation at Ser 276 by PKAc. However, little is known about the formation and activation of the PKAc/AKIP1/p65 complex and the rate at which it enters the nucleus. Initially, we found that the AKIP1 isoform (AKIP 1A) simultaneously binds PKAc and p65 in resting and serum starved cells. Using peptide arrays, we refined the region of AKIP 1A binding on PKAc and mapped the non-overlapping regions on AKIP 1A where PKAc and p65 bind. A peptide to the amino-terminus of PKAc (CAT 1-29) was generated to specifically disrupt the interaction between AKIP 1A and PKAc to study nuclear import of the complex. The rate of p65 nuclear translocation was monitored in the presence or absence of overexpressed AKIP 1A and/or (CAT 1-29). Enhanced nuclear translocation of p65 was observed in the presence of overexpressed AKIP1 and/or CAT 1-29 in cells stimulated with TNFα, and this correlated with decreased phosphorylation of serine 276. To determine whether PKAc phosphorylation of p65 in the cytosol regulated nuclear translocation, serine 276 was mutated to alanine or aspartic acid. Accelerated nuclear accumulation of p65 was observed in the alanine mutant, while the aspartic acid mutation displayed slowed nuclear translocation kinetics. In addition, enhanced nuclear translocation of p65 was observed when PKAc was knocked-down by siRNA. Taken together, these results suggest that AKIP 1A acts to scaffold PKAc to NF-κB in the cytosol by protecting the phosphorylation site and thereby regulating the rate of nuclear translocation of p65.

A kinase interacting protein (AKIP1) is a key regulator of cardiac stress.

Significance Early signaling events leading to protection in the heart under cardiac injury are poorly understood. We identified one such protein, A kinase interacting protein (AKIP1), as a modulator that responds to oxidative stress; up-regulation of AKIP1 showed protection to ischemic injury through enhanced mitochondrial integrity. We show AKIP1 functions as a molecular scaffold via interaction with mitochondrial apoptosis inducing factor and increases protein kinase A activity. These mitochondrial signaling complexes assembled by AKIP1 alter the physiological response of the heart under ischemic stress. Understanding molecular activity and regulation of AKIP1 could lead to novel therapeutic approaches to limit myocardial injury. cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism, and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrial-dependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 gene-transferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release, and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart.

Isoform-specific targeting of PKA to multivesicular bodies

PKA RIα subunit is localized to MVBs by the A-kinase–anchoring protein AKAP11 when disassociated from the PKA catalytic subunit.

Sub-mitochondrial localization of the genetic-tagged mitochondrial intermembrane space-bridging components Mic19, Mic60 and Sam50

ABSTRACT Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space-bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs). Tagged Mic19 and Mic60 were located at CJs, distributed in a network pattern along the mitochondrial periphery and also enriched inside cristae. We discovered an association of Mic19 with cytochrome c oxidase subunit IV. It was also found that tagged Sam50 is not uniformly distributed in the outer mitochondrial membrane and appears to incompletely overlap with Mic19- or Mic60-positive domains, most notably at the CJs. Highlighted Article: By using the novel genetic labels miniSOG and APEX2 combined with electron tomography, the sub-compartmental locations of Mic19, Mic60 and Sam50 were deciphered, which helps determine their physiological function and interaction partners.

Fifty Years Since the Discovery of PKA

In 1968, after Sutherlands discovery of cAMP, Krebs discovered cAMP-dependent Protein Kinase (PKA), thus establishing the major paradigm for cAMP signaling in mammalian cells. The protein kinases, one of the largest gene families, are associated with many diseases, and PKA serves as a prototype for general protein kinase structure and function. While the structure of PKA first defined the conserved protein kinase fold, the recent solution of inhibited complexes of the catalytic (C) and regulatory (R) subunits have defined not only the molecular basis for inhibition but also the mechanisms for activation by cAMP. In addition, they show the dramatic flexibility that is embedded within the R-subunit as it releases cAMP and wraps itself around the large lobe of the C-subunit. In contrast, the C-subunit functions as a stable scaffold that binds to many proteins. In addition to the R-subunit isoforms and PKI, the C-subunit docks to other proteins such as the A Kinase Interacting Protein (AKIP-1), which contributes to its nuclear localization. The R- and C-subunits do not typically exist as isolated entities in the cell but instead are part of large complexes assembled in part through A Kinase Anchoring Proteins (AKAPs). Localization and trafficking are an essential part of kinase function, and the challenge for the next decade will be to understand how these large PKA-mediated signaling complexes assemble and integrate highly regulated functional responses. It is a challenge that requires the concerted integration of structural biology, cell biology, pharmacology and physiology. (Funded by grants from the NIH to SST and fellowships from the American Cancer Society to CK and CWE, and NIH Training Grant GM08326 to CYC.).