Numerous distinct PKA-, or EPAC-based, signalling complexes allow selective phosphodiesterase 3 and phosphodiesterase 4 coordination of cell adhesion
Introduction
Cellular signalling is a complex process requiring the coordinated regulation of a multiplicity of cellular proteins and intracellular chemical messengers. In the context of cAMP signalling, regulation of the synthetic arm of this system coordinates the rate of cAMP synthesis by adenylyl cyclases [ACs] [1]. In several cell types cAMP activates both Protein Kinases A (PKA) and members of a relatively newly described family of cAMP effectors, the Exchange Proteins Activated by cAMP (EPAC). For PKA, binding of cAMP to defined site within both regulatory subunits of the tetrameric holoenzyme allows release and activation of two catalytic (C) subunits and substrate phosphorylation [2]. Activation of EPACs occurs through relief of an intermolecular auto-inhibition following cAMP binding to amino-terminal cAMP binding site(s)[3]. EPACs activate variants of the Rap family of G-proteins and as such act to regulate the activity of Rap-effector enzymes [4]. Activation of PKA and/or EPAC allows cAMP to coordinate myriad events in cells including metabolism, migration, proliferation and adhesion [4].
Selective hydrolysis of the 3′-5′ phosphodiester bond of cAMP by cyclic nucleotide phosphodiesterases (PDEs) catalyzes its inactivation. The PDE super-family consists of eleven distinct families (PDE1-PDE11) of mammalian genes which, in total, can encode greater than fifty distinct enzymes [5], [6]. To date, variants of the PDE3 and PDE4 families of enzymes have received the most study. Although genes encoding PDE3 enzymes were initially cloned from human heart (PDE3A), or from rat adipocytes (PDE3B), respectively [7], [8], it is now clear that PDE3A and PDE3B are each expressed in numerous tissues and cell types. PDE3A encodes three distinct enzymes, namely PDE3A1,-2,-3, and PDE3B encodes only one PDE3B enzyme [9]. PDE3s preferentially catalyze the degradation of cAMP, over cGMP, with the latter competitively inhibiting cAMP hydrolysis by this PDE [10], [11]. In addition to cGMP-based inhibition, PDE3A and PDE3B activities are each stimulated subsequent to PKA- or PKB-mediated phosphorylation of these enzymes at distinct sites [12]. The PDE4 family is comprised of 4 genes [PDE4A–PDE4D] and an extensive literature describes how differential promoter usage, as well as alternate mRNA splicing, coordinate the expression of as many as 16 distinct PDE4 enzymes in cells [13]. In addition, the catalytic activity of several PDE4 variants is increased subsequent to PKA phosphorylation at sites within the upstream conserved region 1 [UCR1] [14], or decreased, through ERK (extracellular-signal-related protein kinase) phosphorylation of a catalytic domain residue [15].
Significant advances have been made during the past few decades in elucidating the molecular mechanisms through which cAMP can act to simultaneously, yet specifically, regulate several effects in cells. Indeed, while early models proposed that cAMP was free to diffuse and act throughout the cell, it is now widely accepted that cAMP can also act locally at sites within cells where cAMP-effector molecules and either adenylyl cyclases, or PDEs, are co-localized. Indeed, recent cell biological and biochemical studies have shown that each PKA and EPACs can be targeted to selective regions of cells and that cAMP can act selectively at these sites by accessing these targeted effectors [16], [17], [18], [19], [20], [21], [22], [23], [24]. For example, using Fluorescence Resonance Energy Transfer [FRET]-based probes which mimic the cAMP binding characteristics of either PKA, or EPAC, several laboratories have shown that cellular cAMP concentrations are non-uniform and that subcellular compartments containing either PKA, or EPAC, or both effectors exist [16], [17], [18], [19], [20], [21], [22], [23], [24]. To date almost all reports of PDE co-localization with cAMP effector proteins has involved PDE4 family enzymes and the generality of this process to members of the other PDE families remains to be established.
In this work, we report that the PDE3B and PDE4D enzymes expressed in HEK 293T cells (herein “293T” cells) are integrated into multiple, non-overlapping, signalling complexes with either PKA or EPAC. In addition, we report that these multiple PDE-containing complexes are involved in coordinating the effect of cAMP-elevating agents on integrin-dependent adhesion.
Section snippets
Materials
293T cells were a kind gift from Dr. Peter Greer (Kingston, ON, Canada). An expression plasmid encoding Vesicular Stomatitis Virus (VSV)-tagged HSPDE4D3 was a gift from Dr. Miles Houslay (Glasgow, Scotland, UK). A plasmid encoding a full-length contiguous cDNA for HSPDE3B was provided by Dr. V.C. Manganiello (Bethesda, MD, USA) and that containing FLAG–EPAC1 was a gift from Dr. X. Cheng (Galveston, TX, USA). pCMV tag 2A, B and C were from Stratagene (La Jolla, CA, USA). [3H]Adenine, [14
PDE3B and PDE4D variants are each components of cAMP effector-containing signalling complexes in 293T cells
Consistent with previous reports, 293T cell cAMP PDE activity was contributed by PDE3 (20 ± 3% using cilostamide (1 μM), (n = 15)) and PDE4 (70 ± 4% using 10 μM Ro, 20–1724, (n = 15)), respectively. Immunoblot analysis of 293T cell lysates identified PDE3B (Fig. 1A(b), upper panel), but not PDE3A (not shown), and potentially several PDE4D variants (Fig. 1A(c), upper panel).
PKA-, and EPAC-containing, cellular complexes were isolated together from 293T cell lysates using the cAMP–agarose adsorption
Discussion
The realization that cAMP simultaneously and selectively regulates myriad distinct cellular events has long represented a signalling puzzle from which subcellular compartmentation offered a mechanistically attractive refuge. Although this explanation has not been universally accepted, recent reports have significantly broadened its general appeal. Thus, several have reported that certain drugs and/or hormones can regulate cAMP levels dynamically within distinct subcellular domains in several
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