AKAP7 Regulates CaM Kinase Activation in MCF-7 Cells

Abstract Estrogen (E2) activates calcium/calmodulin-dependent protein kinases (CaM Kinases) in MCF-7 breast cancer cells. In particular E2 activates a CaM KK, CaM KI, and ERK pathway to promote proliferation. CaM Kinase activation of ERK may be blocked by PKA in certain cell types through direct phosphorylation and inhibition of CaM KK. The ability of PKA to phosphorylate its cellular targets may be dictated by protein kinase A anchoring proteins (AKAPs). Hormones that elevate cAMP and activate PKA may utilize AKAPs to regulate signal transduction. Our goal was to evaluate the role of AKAPs in regulating E2 activation of the CaM KK and CaM KI pathway in MCF-7 cells. We also examined the ability of vitamin D (VitD) working through cAMP and PKA to block CaM KK signaling in breast cancer cells. Our results suggest that E2 activates CaM KK and CaM KI within 5 minutes. VitD promoted PKA-dependent phosphorylation of CaM KK. VitD and epinephrine treatment of cells triggered a potent increase in cAMP levels. Interestingly, purified GST-RII pulled down both CaM KK and CaM KI. Similarly, purified AKAP7 but not AKAP5 bound CaM KK and CaM KI an effect that is enhanced with E2. Endogenous AKAP7 and CaM KK associated in E2-stimulated but not in VitD-treated cells and VitD also blocked CaM KK activation in MCF-7 cells. Our results suggest that VitD blocks E2 activation of CaM Kinases and their association with AKAP7 in MCF-7 cells

LKB1 Destabilizes Microtubules in Myoblasts and Contributes to Myoblast Differentiation

Background: Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified. Findings: We find that a transient decrease in microtubule stabilization early during myoblast differentiation precedes the ultimate microtubule stabilization seen in differentiated myotubes. We report a role for the serine-threonine kinase LKB1 in both microtubule destabilization and myoblast differentiation. LKB1 overexpression reduced microtubule elongation in a Nocodazole washout assay, and LKB1 RNAi increased it, showing LKB1 destabilizes microtubule assembly in myoblasts. LKB1 levels and activity increased during myoblast differentiation, along with activation of the known LKB1 substrates AMPactivated protein kinase (AMPK) and microtubule affinity regulating kinases (MARKs). LKB1 overexpression accelerated differentiation, whereas RNAi impaired it. Conclusions: Reduced microtubule stability precedes myoblast differentiation and the associated ultimate microtubule stabilization seen in myotubes. LKB1 plays a positive role in microtubule destabilization in myoblasts and in myoblast differentiation. This work suggests a model by which LKB1-induced microtubule destabilization facilitates the cytoskeleta

The mAKAP signaling complex: Integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G s-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones

Signalosomes as therapeutic targets

Cardiac hypertrophy is the predominant compensatory response of the heart to a wide variety of biomechanical stressors, including exercise, hypertension, myocardial infarction, intrinsic cardiomyopathy or congenital heart disease. Although cardiac hypertrophy can maintain cardiac output in response to elevated wall stress, sustained cardiac hypertrophy is often accompanied by maladaptive remodeling which can ultimately lead to heart failure. Cultured cardiac myocytes, transgenic and knock-out animal models, and pharmacological studies have not only revealed key molecules involved in hypertrophic signaling, but have also highlighted the redundancy in the hypertrophic signaling cascade. Currently, the majority of existing therapies for inhibition of pathologic cardiac hypertrophy and heart failure target molecules on the surface of cardiac myocytes, such as G-protein coupled receptors (GPCRs) and ion channels. Because these molecules are upstream of multiple intracellular signaling pathways, however, current therapy is often accompanied by significant off-target effects and toxicity. More recently, research has focused on identifying the intracellular effectors of these signaling cascades in the hope that more selective drugs may be rationally designed for therapeutic intervention. Within the cardiac myocyte, the formation of discrete multimolecular complexes, or ‘signalosomes’, is an important mechanism for increasing the specificity and efficiency of hypertrophic signal transduction. In response to extracellular stimuli, these signalosomes can alter gene and protein expression, cell size, and chamber remodeling, such as in the case of the signalosomes formed by the mAKAPβ and AKAP-lbc scaffold proteins. A better understanding of the basic molecular mechanisms regulating the compartmentation and scaffolding of signaling molecules could lead to the development of new clinical tools that may prevent the development of heart failure and minimize negative impacts on physiological processes

mAKAPβ signalosomes - A nodal regulator of gene transcription associated with pathological cardiac remodeling.

Striated myocytes compose about half of the cells of the heart, while contributing the majority of the heart's mass and volume. In response to increased demands for pumping power, including in diseases of pressure and volume overload, the contractile myocytes undergo non-mitotic growth, resulting in increased heart mass, i.e. cardiac hypertrophy. Myocyte hypertrophy is induced by a change in the gene expression program driven by the altered activity of transcription factors and co-repressor and co-activator chromatin-associated proteins. These gene regulatory proteins are subject to diverse post-translational modifications and serve as nuclear effectors for intracellular signal transduction pathways, including those controlled by cyclic nucleotides and calcium ion. Scaffold proteins contribute to the underlying architecture of intracellular signaling networks by targeting signaling enzymes to discrete intracellular compartments, providing specificity to the regulation of downstream effectors, including those regulating gene expression. Muscle A-kinase anchoring protein β (mAKAPβ) is a well-characterized scaffold protein that contributes to the regulation of pathological cardiac hypertrophy. In this review, we discuss the mechanisms how this prototypical scaffold protein organizes signalosomes responsible for the regulation of class IIa histone deacetylases and cardiac transcription factors such as NFAT, MEF2, and HIF-1α, as well as how this signalosome represents a novel therapeutic target for the prevention or treatment of heart failure

AKAPs: The architectural underpinnings of local cAMP signaling

The cAMP-dependent protein kinase A (PKA) is targeted to specific compartments in the cardiac myocyte by A-kinase anchoring proteins (AKAPs), a diverse set of scaffold proteins that have been implicated in the regulation of excitation–contraction coupling and cardiac remodeling. AKAPs bind not only PKA, but also a large variety of structural and signaling molecules. In this review, we discuss the basic concepts underlying compartmentation of cAMP and PKA signaling, as well as a few of the individual AKAPs that have been shown to be functionally relevant in the heart. This article is part of a Special Issue entitled "Local Signaling in Myocytes". ► In this review, we discuss A-kinase anchoring proteins expressed in the heart. ► AKAPs are important for cAMP compartmentation. ► AKAP scaffold proteins confer specificity and fidelity to cAMP signaling

The Large Isoforms of A-Kinase Anchoring Protein 18 Mediate the Phosphorylation of Inhibitor-1 by Protein Kinase A and the Inhibition of Protein Phosphatase 1 ActivityS⃞

Inhibitor-1 (I-1) is phosphorylated on threonine residue 35 (Thr35) by the cAMP-dependent protein kinase (PKA), inducing the potent inhibition of the serine-threonine-specific protein phosphatase 1 (PP1). We now report that the formation of a signaling complex containing PKA and I-1 by the A-kinase anchoring protein 18 (AKAP18) facilitates this regulation in cells. AKAP18 directly bound I-1, and AKAP18/I-1 complexes were isolated from both rat heart extract and transfected heterologous cells. It is noteworthy that prevention of PKA binding to the AKAP18 scaffold decreased I-1 phosphorylation by 48% in cells. Moreover, the I-1 target PP1 was also associated with AKAP18 complexes. The cAMP-mediated inhibition of phosphatase activity was contingent on PKA binding to the scaffold. These observations reveal an additional level of complexity in PP1 regulation because of its association with AKAP18 multimolecular signaling complexes and suggest that targeting of AKAP18 complexes may be an alternative method to alter phosphatase activity and modulate specific substrate dephosphorylation