Stefan Strack

Reversible phosphorylation of Drp1 by cyclic AMP‐dependent protein kinase and calcineurin regulates mitochondrial fission and cell death

Opposing mitochondrial fission and fusion reactions determine the shape and interconnectivity of mitochondria. Dynamin‐related protein 1 (Drp1) is an ancient mechanoenzyme that uses GTP hydrolysis to power the constriction and division of mitochondria. Although Drp1‐mediated mitochondrial fragmentation is recognized as an early event in the apoptotic programme, acute regulation of Drp1 activity is poorly understood. Here, we identify a crucial phosphorylation site that is conserved in all metazoan Drp1 orthologues. Ser 656 is phosphorylated by cyclic AMP‐dependent protein kinase and dephosphorylated by calcineurin, and its phosphorylation state is controlled by sympathetic tone, calcium levels and cell viability. Pseudophosphorylation of Drp1 by mutation of Ser 656 to aspartic acid leads to the elongation of mitochondria and confers resistance to various pro‐apoptotic insults. Conversely, the constitutively dephosphorylated Ser656Ala mutant Drp1 promotes mitochondrial fragmentation and increases cell vulnerability. Thus, Drp1 phosphorylation at Ser 656 provides a mechanism for the integration of cAMP and calcium signals in the control of mitochondrial shape, apoptosis and other aspects of mitochondrial function.

AUTOPHOSPHORYLATION-DEPENDENT TARGETING OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II BY THE NR2B SUBUNIT OF THE N-METHYL-D-ASPARTATE RECEPTOR

Activation and Thr286autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx viaN-methyl-d-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII·NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca2+influx, and Thr286 autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca2+channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation.

Mechanism and Regulation of Calcium/Calmodulin-dependent Protein Kinase II Targeting to the NR2B Subunit of the N-Methyl-D-aspartate Receptor*

Calcium influx through theN-methyl-d-aspartate (NMDA)-type glutamate receptor and activation of calcium/calmodulin-dependent kinase II (CaMKII) are critical events in certain forms of synaptic plasticity. We have previously shown that autophosphorylation of CaMKII induces high-affinity binding to the NR2B subunit of the NMDA receptor (Strack, S., and Colbran, R. J. (1998) J. Biol. Chem. 273, 20689–20692). Here, we show that residues 1290–1309 in the cytosolic tail of NR2B are critical for CaMKII binding and identify by site-directed mutagenesis several key residues (Lys1292, Leu1298, Arg1299, Arg1300, Gln1301, and Ser1303). Phosphorylation of NR2B at Ser1303 by CaMKII inhibits binding and promotes slow dissociation of preformed CaMKII·NR2B complexes. Peptide competition studies imply a role for the CaMKII catalytic domain, but not the substrate-binding pocket, in the association with NR2B. However, analysis of monomeric CaMKII mutants indicates that the holoenzyme structure may also be important for stable association with NR2B. Residues 1260–1316 of NR2B are sufficient to direct the subcellular localization of CaMKII in intact cells and to confer dynamic regulation by calcium influx. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B bidirectionally modulates colocalization with CaMKII after NMDA receptor activation, suggesting a dynamic model for the translocation of CaMKII to postsynaptic targets.

Differential inactivation of postsynaptic density-associated and soluble Ca2+/calmodulin-dependent protein kinase II by protein phosphatases 1 and 2A.

Abstract: Autophosphorylation of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) at Thr286 generates Ca2+‐independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr286 in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A‐like enzymes account for >70% of activity toward exogenous soluble Thr286‐autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr286‐autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (32P autophosphorylated in the presence of Ca2+/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD‐associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD‐associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca2+‐independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.

Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins

The serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 A resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11-15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.

CaMKII determines mitochondrial stress responses in heart

Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU) are not known. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced IMCU and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.

Brain protein phosphatase 2A: Developmental regulation and distinct cellular and subcellular localization by B subunits

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B). We have investigated the spatial and temporal expression patterns of three members of the B subunit family, Bα, Bβ, and Bγ, both at the message level by using ribonuclease protection analysis and at the protein level by using specific antibodies. Although A, Bα, and C protein are expressed in many tissues, Bβ and Bγ were detectable only in brain. Bα, Bβ, and Bγ are components of the brain PP2A heterotrimer, because they copurified with A and C subunits on immobilized microcystin. Whereas Bα and Bβ are mainly cytosolic, Bγ is enriched in the cytoskeletal fraction. In contrast to A, C, and Bα, which are expressed at constant levels, Bβ and Bγ RNA and protein are developmentally regulated, with Bβ levels decreasing and Bγ levels increasing sharply after birth. RNA and immunoblot analyses of subdissected brain regions as well as immunohistochemistry demonstrated that B subunits are expressed in distinct but overlapping neuronal populations and cellular domains. These data indicate that B subunits confer tissue and cell specificity, subcellular localization, and developmental regulation to the PP2A holoenzyme. The Bα‐containing heterotrimer may be important in general neuronal functions that involve its partially nuclear localization. Holoenzymes containing Bβ likely function in early brain development as well as in somata and processes of subsets of mature neurons. Bγ may target PP2A to cytoskeletal substrates that are important in the establishment and maintenance of neuronal connections. J. Comp. Neurol. 392:515–527, 1998.

Protein kinase A anchoring via AKAP150 is essential for TRPV1 modulation by forskolin and prostaglandin E2 in mouse sensory neurons

Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E2 (PGE2). However, little is known about the molecular organization of the TRPV1 phosphorylation complex and specifically about scaffolding proteins that position the protein kinase A (PKA) holoenzyme proximal to TRPV1 for effective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in ∼80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE2 decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE2 effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (Δ36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in Δ36 mice. The PGE2/PKA signaling defect in DRG neurons from Δ36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE2-induced thermal hyperalgesia was significantly diminished in Δ36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE2/PKA signaling pathway.

Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1.

The mitochondrial signaling complex PKA/AKAP1 protects neurons against mitochondrial fragmentation and cell death by phosphorylating and inactivating the mitochondrial fission enzyme Drp1.

Positive Regulation of Raf1-MEK1/2-ERK1/2 Signaling by Protein Serine/Threonine Phosphatase 2A Holoenzymes

Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Bα/δ regulatory subunits by ∼3-fold or knock-down of Bα by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Bα/δ subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Bα knock-down cells but elevated in Bα- and Bδ-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Bα or Bδ. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABαC and ABδC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABαC and ABδC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.