An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria

Abstract

A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10-100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925-949 and 1,140-1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pulldown photobleaching experiments show that 41 AE 10% of SKIP sequesters two YFP-RI dimers, whereas 59 AE 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.

Targeting cAMP/PKA pathway for glycemic control and type 2 diabetes therapy

“…It should be noted that AKAP150 binds type II-PKA (RIIα and RIIβ) with much higher affinity than RIα (Herberg et al 2000), suggesting that, in addition to RIα-PKA (Hussain et al 2012), type II PKA may also be involved in the process of insulin secretion. Although RIα is usually considered soluble, dual-specific AKAPs (Jarnaess et al 2008) that bind both RI and RII subunits and RI-specific AKAPs (Means et al 2011, Burgers et al 2012) have been identified. It remains unknown if such an AKAP exists in pancreatic β cells to mediate the regulation of insulin secretion by RIα-PKA.…”

Local cAMP signaling in disease at a glance

“…AKAP-PKA complexes are directed to discrete subcellular locations by further protein-protein or protein-lipid interactions with the anchoring proteins (Wong and Scott, 2004). The majority of AKAPs selectively bind the RII subunits of PKAs, with a few exceptions such as the RI-selective AKAP sphingosine kinase interacting protein (SKIP) (Means et al, 2011). Accordingly, type I PKA holoenzymes exhibit a more homogeneous and cytoplasmic distribution than the type II PKA holoenzymes that remain tethered via AKAPs to a variety of cell membranes and intracellular organelles (Wong and Scott, 2004).…”

Bringing single-molecule spectroscopy to macromolecular protein complexes

“…The researchers have also reported proof-of-principle studies using systems such as mTOR (mammalian target of rapamycin) protein complexes and membrane receptor proteins [17]. The same group has extended its research to stoichiometry analyses of AKAPs (A-kinase anchoring proteins) [48], ORCA (origin-recognition complex-associated) protein complexes [49], and peptide loading complexes [50].…”

“…The researchers have also reported proof-of-principle studies using systems such as mTOR (mammalian target of rapamycin) protein complexes and membrane receptor proteins [17]. The same group has extended its research to stoichiometry analyses of AKAPs (A-kinase anchoring proteins) [48], ORCA (origin-recognition complex-associated) protein complexes [49], and peptide loading complexes [50].Jain et al also demonstrated the analysis of endogenous proteins obtained from mouse brain and heart tissues ( Figure 4c). Although it is often demanding to analyze endogenous proteins due to their low abundance, this difficulty can be overcome by the high sensitivity of singlemolecule complex analysis.…”