Structural Basis for the Regulation of Protein Kinase A by Activation Loop Phosphorylation

Steichen, Jon M.; Kuchinskas, Michael; Keshwani, Malik M.; Yang, Jie; Adams, Joseph A.; Taylor, Susan S.

doi:10.1074/jbc.m111.335091

Cited by 80 publications

(92 citation statements)

References 31 publications

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“…This highly conserved site can be found in the activation loop of PKAc subunit as well (Thr197). In both cases, autophosphorylation has been confirmed upon kinase activation (42,43). In Fig.…”

Section: Pka Rii Subunits Form Protein Complexes With Rac1 In Vitro Amentioning

confidence: 57%

Reciprocal regulation of PKA and Rac signaling

Bachmann

Riml

Huber

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac-bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTPRac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA-boosted activation of Raf-Mek-Erk. Consequently GTPRac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac-mediated activation of nuclear Erk1/2 signaling. signal transduction | cross-talk S ignal transduction cascades coordinate the plethora of extracellular stimuli into biological responses within cells. The specificity of receptor-initiated signaling responses is encoded by spatial and temporal dynamics of downstream signaling networks (1). These networks, initiating from e.g., the G protein-coupled receptor (GPCR) superfamily and receptor tyrosine kinases (RTK), tightly regulate signaling pathways at several critical points via feedback loops and cross-talk among other pathways (2-5). A large number of GPCR signaling cascades uses cAMP as an intracellular second messenger (3, 6). In response to hormone binding to distinct GPCRs, cAMP is produced and binds to its canonical effector, the cAMP-dependent protein kinase A (PKA). cAMP binding to the PKA regulatory subunits (R) induces dissociation of the tetrameric PKA holoenzyme, resulting in active PKA catalytic subunits (PKAc; Fig. 1C) (7, 8). To ensure substrate specificity, PKA is tethered to distinct subcellular compartments through physical interaction with A-kinase anchoring proteins (AKAPs; refs. 9 and 10). It has been long regarded that the...

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Section: Pka Rii Subunits Form Protein Complexes With Rac1 In Vitro Amentioning

confidence: 57%

Reciprocal regulation of PKA and Rac signaling

Bachmann

Riml

Huber

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Indeed, the HRD-Asp is one of the most highly conserved residues in the protein kinase superfamily and mutation of the HRD-Asp abolishes catalytic activity in many protein kinases (20,22,23). Unlike the HRD-Asp, the arginine (Arg) within the HRD motif is conserved in most but not all kinases, and plays a largely regulatory role (9,22,24,25). In kinases that are regulated by phosphorylation of the substrate binding activation loop (Fig.…”

Section: Ramachandran Plotmentioning

confidence: 99%

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

Oruganty¹,

Talathi²,

Wood³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The protein kinase catalytic domain contains several conserved residues of unknown functions. Here, using a combination of computational and experimental approaches, we show that the function of some of these residues is to maintain the backbone geometry of the active site in a strained conformation. Specifically, we find that the backbone geometry of the catalytically important HRD motif deviates from ideality in high-resolution structures and the strained geometry results in favorable hydrogen bonds with conserved noncatalytic residues in the active site. In particular, a conserved aspartate in the F-helix hydrogen bonds to the strained HRD backbone in diverse eukaryotic and eukaryotic-like protein kinase crystal structures. Mutations that alter this hydrogen-bonding interaction impair catalytic activity in Aurora kinase. Although the backbone strain is present in most active conformations, several inactive conformations lack the strain because of a peptide flip in the HRD backbone. The peptide flip is correlated with loss of hydrogen bonds with the F-helix aspartate as well as with other interactions associated with kinase regulation. Within protein kinases that are regulated by activation loop phosphorylation, the strained residue is an arginine, which coordinates with the activation loop phosphate. Based on analysis of strain across the protein kinase superfamily, we propose a model in which backbone strain co-evolved with conserved residues for allosteric control of catalytic activity. Our studies provide new clues for the design of allosteric protein kinase inhibitors.allostery | conformational strain | DFG flip | hydrophobic spine | Ramachandran plot

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“…For maximal activity, each catalytic subunit must also be either autophosphorylated or phosphorylated at Thr197, which helps orient catalytic residues in the active site (Steichen et al ., 2010, 2012). However, knowledge of PKA's other posttranslational modification remains limited.…”

Section: Introductionmentioning

confidence: 99%

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Xie

Jin

et al. 2016

Aging Cell

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SummaryAlzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.

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Structural Basis for the Regulation of Protein Kinase A by Activation Loop Phosphorylation

Cited by 80 publications

References 31 publications

Reciprocal regulation of PKA and Rac signaling

Reciprocal regulation of PKA and Rac signaling

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

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