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.

Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.

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.

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)1,2. However, the signaling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU)3–5 are not known. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is activated in ischemia reperfusion (I/R), myocardial infarction (MI) and neurohumoral injury, common causes of myocardial death and heart failure, suggesting 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 (CsA), an mPTP antagonist with clinical efficacy in I/R injury6, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to I/R injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition are resistant to I/R injury, MI and neurohumoral injury, suggesting pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry and suggest mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure dysfunction in response to common experimental forms of pathophysiological stress.

Calcium influx through the N-methyl-D-aspartate CaMKII1 is a family of ubiquitous, calcium/calmodulin-dependent kinases with broad substrate specificity (1). The ␣ and ␤ isoforms are especially abundant in brain, constituting as much as 2% of total protein in the hippocampus (2). There is now overwhelming evidence that CaMKII is central to the mechanism of hippocampal, NMDA receptor-dependent longterm potentiation (LTP), a widely studied cellular model of learning and memory. Reduction of CaMKII activity by pharmacological or genetic means impairs LTP (3), whereas injecting or overexpressing CaMKII increases synaptic strength, which occludes and is occluded by electrically induced LTP (4, 5). Crucial to its function in LTP and spatial learning (6, 7), CaMKII undergoes rapid autophosphorylation following NMDA receptor-mediated calcium influx at a specific residue in its autoregulatory domain (Thr 286 in the ␣ isoform of CaMKII). This autophosphorylation renders the kinase calcium-independent and has been proposed as a form of molecular memory (8). In support, recent in vitro studies show that CaMKII autophosphorylation permits integration of oscillating calcium signals (9).We have recently demonstrated a second role for Thr 286 autophosphorylation, namely in promoting translocation of CaMKII to postsynaptic densities (PSDs) (10), cytoskeletal scaffolds for the neurotransmitter receptor, ion channels, and their regulators. The search for proteins that target Thr 286 -autophosphorylated CaMKII ([P-T286]CaMKII) to the PSD initially identified a 190-kDa binding activity (11), corresponding in size to the highly PSD-enriched NR2A and NR2B subunits of the NMDA receptor. Indeed, we recently showed that NR2B is a binding protein for [P-T286]CaMKII and isolated a CaMKII⅐NMDA receptor complex from PSDs (12). Subsequently, other laboratories implicated NR2A and NR2B (13,14) and NR1 and NR2B (15) as CaMKII-binding proteins. In this report, we identify amino acids critical for CaMKII binding in NR2B and investigate the regulation of CaMKII targeting to NR2B in vitro and in cells. EXPERIMENTAL PROCEDURES MaterialsThe phospho-Thr 286 -specific CaMKII antibody was a generous gift from Said Goueli (Promega). The recombinant ␥1 isoform of the protein phosphatase 1 catalytic subunit was generously provided by Dr. E. Lee (New York Medical College, Valhalla, NY). Sources of other materials are indicated below. Construction, Mutagenesis, and Expression of cDNAsNR2B Mutants-Fragments of the rat NR2B cDNA were amplified by polymerase chain reaction (PCR) with primers incorporating 5Ј-BamHI and 3Ј-EcoRI sites and subcloned into pGEX-2T (Amersham Pharmacia Biotech) for expression of glutathione S-transferase (GST) fusion proteins. All NR2B mutants were generated in the context of the NR2B-(1260 -1339) sequence, which includes the core CaMKII-binding domain (see Fig. 2) flanked by AlwNI and NdeI sites, allowing for single-step ligation of the mutagenized fragment into the full-length