Deposition of fibrillar amyloid ␤ (fA␤) plays a critical role in Alzheimer's disease (AD). We have shown recently that fA␤-induced dystrophy requires the activation of focal adhesion proteins and the formation of aberrant focal adhesion structures, suggesting the activation of a mechanism of maladaptative plasticity in AD. Focal adhesions are actin-based structures that provide a structural link between the extracellular matrix and the cytoskeleton. To gain additional insight in the molecular mechanism of neuronal degeneration in AD, here we explored the involvement of LIM kinase 1 (LIMK1), actin-depolymerizing factor (ADF), and cofilin in A␤-induced dystrophy. ADF/cofilin are actin-binding proteins that play a central role in actin filament dynamics, and LIMK1 is the kinase that phosphorylates and thereby inhibits ADF/cofilin. Our data indicate that treatment of hippocampal neurons with fA␤ increases the level of Ser3-phosphorylated ADF/cofilin and Thr508-phosphorylated LIMK1 (P-LIMK1), accompanied by a dramatic remodeling of actin filaments, neuritic dystrophy, and neuronal cell death. A synthetic peptide, S3 peptide, which acts as a specific competitor for ADF/cofilin phosphorylation by LIMK1, inhibited fA␤-induced ADF/cofilin phosphorylation, preventing actin filament remodeling and neuronal degeneration, indicating the involvement of LIMK1 in A␤-induced neuronal degeneration in vitro. Immunofluorescence analysis of AD brain showed a significant increase in the number of P-LIMK1-positive neurons in areas affected with AD pathology. P-LIMK1-positive neurons also showed early signs of AD pathology, such as intracellular A␤ and pretangle phosphorylated tau. Thus, LIMK1 activation may play a key role in AD pathology.

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein ␤ (mAKAP␤) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAP␤ selectively binds type 5 AC in the heart and that mAKAP␤-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAP␤-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAP␤-AC association. Accordingly, disruption of mAKAP␤-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAP␤ complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.The formation of multimolecular protein complexes contributes to the specificity of intracellular signaling pathways, including those regulating cardiac myocyte hypertrophy. The cAMP-dependent protein kinase (PKA) 3 is targeted to specific intracellular domains by protein kinase A-anchoring proteins (AKAPs) that often serve as scaffolding proteins for diverse signaling enzymes (1). In the heart, global disruption of PKA anchoring affects cardiac contractility, while the inhibited expression of individual AKAPs such as mAKAP␤ or AKAPLbc attenuates adrenergic-induced hypertrophy of cultured neonatal myocytes (2-4). We have recently shown that specific AKAPs, namely AKAP79 and Yotiao, bind adenylyl cyclases (AC) (5, 6). However, the functional significance of AC-AKAP complexes has not been demonstrated. mAKAP␤, expressed in striated myocytes, is one of two known splice variants encoded by the single mAKAP (AKAP6) gene (7). We previously published that mAKAP␤ is primarily localized to the outer membrane of the nuclear envelope via direct binding to nesprin-1␣ (4, 8). In cardiac myocytes, mAKAP␤ serves as the scaffold for a multimolecular signaling complex that in addition to PKA includes the ryanodine receptor (RyR2), the protein phosphatases PP2A and calcineurin, phosphodiesterase 4D3 (PDE4D3), exchange protein activated by cAMP (Epac1), ERK5, and MEK5 mitogen-activated protein kinases, molecules implicated in the regulation of cardiac hypertrophy (4, 7-13). mAKAP␤ complexes facilitate crosstalk between MAP kinase, calcium, and cAMP signaling pathways, permitting feedback inhibition of cAMP levels and the dynamic regulation of PKA and ERK5 activity (4, 9 -13). Accordingly, mAKAP␤ RNAi attenuates adrenergic and cytokine-induced hypertrophy of...

The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085-2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56␦. Like PDE4D3, B56␦ is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56␦ enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56␦ mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56␦ phosphorylation. In general, PKA⅐PP2A⅐mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling.

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Amyloid beta (Abeta) is a metabolic product of Abeta precursor protein (APP). Deposition of Abeta in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Abeta induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor-like protein for Abeta fibrils (fAbeta). In this study, we present further experimental support for the direct interaction of APP with fAbeta and for its involvement in Abeta neurotoxicity. Using recombinant purified holo-APP (h-APP), we have shown that it directly binds fAbeta. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAbeta. One sequence in the n-terminus of APP is required for binding of fAbeta to secreted APP (s-APP) but not to h-APP. In addition, the extracellular juxtamembrane Abeta-sequence mediates binding of fAbeta to h-APP but not to s-APP. Deletion of the extracellular juxtamembrane Abeta sequence abolishes abnormal h-APP accumulation and toxicity induced by fAbeta deposition, whereas deletions in the n-terminus of APP do not affect Abeta toxicity. These experiments show that interaction of toxic Abeta species with its membrane-anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Abeta-receptor-like function of APP directly implicated in neuronal degeneration in AD.