WAVE1--the Wiskott-Aldrich syndrome protein (WASP)--family verprolin homologous protein 1--is a key regulator of actin-dependent morphological processes in mammals, through its ability to activate the actin-related protein (Arp2/3) complex. Here we show that WAVE1 is phosphorylated at multiple sites by cyclin-dependent kinase 5 (Cdk5) both in vitro and in intact mouse neurons. Phosphorylation of WAVE1 by Cdk5 inhibits its ability to regulate Arp2/3 complex-dependent actin polymerization. Loss of WAVE1 function in vivo or in cultured neurons results in a decrease in mature dendritic spines. Expression of a dephosphorylation-mimic mutant of WAVE1 reverses this loss of WAVE1 function in spine morphology, but expression of a phosphorylation-mimic mutant does not. Cyclic AMP (cAMP) signalling reduces phosphorylation of the Cdk5 sites in WAVE1, and increases spine density in a WAVE1-dependent manner. Our data suggest that phosphorylation/dephosphorylation of WAVE1 in neurons has an important role in the formation of the filamentous actin cytoskeleton, and thus in the regulation of dendritic spine morphology.

Our previous studies of DARPP-32 in striatal slices have shown that activation of D1 receptors leads to cAMP-dependent dephosphorylation of Thr-75, the Cdk5 site in DARPP-32. In the current study, we have elucidated a mechanism whereby protein phosphatase 2A (PP2A) is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of Thr-75. PP2A consists of a catalytic C subunit that associates with the scaffolding A subunit and a variety of B subunits. We have found that the A/C subunits of PP2A, in association with the B56␦ (or PPP2R5D) regulatory subunit, is an active DARPP-32 phosphatase. The B56␦ subunit expressed in HEK293 cells forms a heterotrimeric assembly that catalyzes PKA-mediated dephosphorylation at Thr-75 in DARPP-32 (also cotransfected into HEK293 cells). The B56␦ subunit is phosphorylated by PKA, and this increases the overall activity of PP2A in vitro and in vivo. Among four PKA-phosphorylation sites identified in B56␦ in vitro, Ser-566 was found to be critical for the regulation of PP2A activity. Moreover, Ser-566 was phosphorylated by PKA in response to activation of D1 receptors in striatal slices. Based on these studies, we propose that the B56␦/A/C PP2A complex regulates the dephosphorylation of DARPP-32 at Thr-75, thereby helping coordinate the efficacy of dopaminergic neurotransmission in striatal neurons. Moreover, stimulation of protein phosphatase activity by this mechanism may represent an important signaling pathway regulated by cAMP in neurons and other types of cell.cAMP ͉ DARPP-32 ͉ protein phosphorylation D ARPP-32 is a phosphoprotein that is highly enriched in dopaminoceptive medium-sized spiny neurons in the striatum and nucleus accumbens (1, 2). A variety of biochemical studies as well as targeted deletion and mutation of DARPP-32 in mice have shown that DARPP-32 plays a critical role in the actions of dopamine as well as in the actions of antipsychotic drugs, drugs of abuse, and other agents that modulate dopamine levels in the brain (2-5). Through activation of the D1 subclass of receptors, dopamine increases cAMP, activates protein kinase A (PKA), and phosphorylates Thr-34 of DARPP-32. Phosphorylation at Thr-34 converts DARPP-32 into a potent, high-affinity inhibitor of the broad specificity serine/threonine protein phosphatase, PP-1, leading to increased phosphorylation of many physiologically important substrates in medium spiny neurons, including neurotransmitter receptors, voltage-gated ion channels, ion pumps, protein kinases, and transcription factors (1, 2).In addition to Thr-34, DARPP-32 is phosphorylated at multiple sites by several protein kinases, including CK1, CK2 and Cdk5 (6-9). In particular, phosphorylation of Thr-75 by Cdk5 blocks PKA-mediated phosphorylation of Thr-34 of DARPP-32, thereby modulating the efficacy of the dopamine/D1/cAMP/ PKA/DARPP-32/PP1 signaling cascade (8). Our previous studies have found that there is a reciprocal relationship between the phosphorylation status of . Under basal conditions in striatal neurons in vivo or in vit...

SummaryDopamine orchestrates motor behavior and reward-driven learning. Perturbations of dopamine signaling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here, we show that drugs of abuse, as well as natural reinforcement learning, promote the nuclear accumulation of dopamine-and cAMPregulated phosphoprotein Mr=32,000 . This accumulation is mediated through a signaling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser-97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser-97 profoundly alters behavioral effects of drugs of abuse, and decreases motivation for food, underlining the functional importance of this signaling cascade.Midbrain dopamine (DA) neurons, activated following unexpected rewarding stimuli, are essential in reinforcement learning 1 . Drugs of abuse mimic the physiological action of DA neurons by increasing their firing rate or preventing DA uptake. Thus, they enhance extracellular DA levels in the forebrain, especially in the nucleus accumbens (NAc), a key structure required for the reinforcing effects of addictive drugs [2][3][4] . To understand how DA mediates reward-controlled learning, it is necessary to identify the intracellular events that trigger gene transcription alterations supporting long-lasting synaptic changes [5][6][7] . DARPP-32 (dopamine-and cAMP-regulated phosphoprotein, Mr=32,000) 8 is a prominent mediator of DA NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript signaling in the striatum 9 . DARPP-32 is highly enriched in striatal GABAergic mediumsize spiny neurons (MSN) 10 . Following activation of DA D1 receptors (D1R), DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) at Thr-34 and converted into a potent inhibitor of the multifunctional serine/threonine protein phosphatase-1 (PP1) 11 . DARPP-32-mediated inhibition of PP1 increases the phosphorylation of neurotransmitter receptors and ion channels crucial for synaptic function and plasticity 9 . DARPP-32 also regulates nuclear events, as demonstrated by alterations of drug-induced gene expression in mice lacking DARPP-32 or bearing a point mutation of 13 . Part of the control exerted by DARPP-32 on transcription is mediated by activation of the ERK pathway, dependent on the concomitant stimulation of D1R and glutamate NMDA receptors 13,14 . However, the precise mechanisms of information transfer from the cytoplasm to the nucleus of striatal neurons are still poorly characterized. Drugs of abuse and reinforcement learning trigger nuclear accumulation of DARPP-32 in striatal neuronsDARPP-32 has been extensively characterized as a cytoplasmic...

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.

SUMMARY p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin-reuptake inhibitors (SSRIs). Here we have identified SMARCA3, a chromatin-remodeling factor, as a novel target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. In response to the SSRI, fluoxetine, the expression of p11 is induced in both cell types, and the amount of the ternary complex of p11/annexin A2/SMARCA3 is increased. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway, and suggest a novel approach to the development of improved antidepressant therapies.