1 Isoform-specific subcellular localization and function of protein kinase A identified by 2 mosaic imaging of mouse brain 3 Ronit Ilouz1*, Varda Lev-Ram1, Eric A. Bushong2, Travis Stiles3, Dinorah Friedmann- 4 Morvinski4,5, Christopher Douglas3, Geoffrey Goldberg3, Mark H. Ellisman2,6, and Susan S. 5 Taylor1,7* 6 7 8 9 10 11 12 13 14 1Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA. 15 2Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of 16 California, San Diego, La Jolla, CA 92093, USA. 17 18 3Department of Ophthalmology, Shiley Eye Center, University of California, San Diego, La Jolla, CA 92093, USA 19 20 4Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA 21 22 5Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel Aviv 23 University, Tel Aviv 69978, Israel 24 6Department of Neurosciences, University of California, San Diego School of Medicine, La Jolla, CA 92093, USA. 25 7Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA 26 27 28 * Correspondence: [email protected], [email protected] 29 1 30 ABSTRACT 31 Protein kinase A (PKA) plays critical roles in neuronal function that are mediated by different 32 regulatory (R) subunits. Deficiency in either RIβ or RIIβ subunits results in distinct neuronal 33 phenotypes. Although RIβ contributes to synaptic plasticity, it is the least studied isoform. Using 34 isoform-specific antibodies we generated high-resolution large-scale immunohistochemical 35 mosaic images of mouse brain that provide global views of several brain regions, including the 36 hippocampus and cerebellum. The isoforms concentrate in discrete brain regions and we then 37 zoom-in to show distinct patterns of subcellular localization. RIβ is enriched in dendrites and co- 38 localizes with MAP2, whereas RIIβ is concentrated in axons. Using correlated light and electron 39 microscopy we confirm mitochondrial and nuclear localization of RIβ in cultured neurons. To 40 show the functional significance of nuclear localization, we demonstrate that down-regulation of 41 RIβ, but not RIIβ, decreased CREB phosphorylation. Our study reveals how PKA isoform 42 specificity is defined by precise localization. 43 44 45 46 47 48 49 2 50 INTRODUCTION 51 Precise spatiotemporal regulation of signaling molecules is central to the intricacies of 52 signal transduction. cAMP-dependent protein kinase (PKA), ubiquitously expressed in every 53 mammalian cell, regulates numerous signaling pathways and is critical for many neuronal 54 functions. This includes learning and memory (Kandel, 2012) and multiple forms of synaptic 55 plasticity (Abel et al., 1997; Yasuda et al., 2003). Pharmacological or genetic inhibition of PKA 56 severely affects the induction of hippocampal long-term potentiation and inhibits synaptic 57 plasticity and long lasting memory (Abel et al., 1997). A reduction in PKA signaling contributes 58 to the etiology of several neurodegenerative diseases, including Alzheimer’s disease and 59 Parkinson’s disease (Dagda and Das Banerjee, 2015; Howells et al., 2000). Phosphorylation 60 mediated by cAMP signaling is critically involved in cell growth, differentiation, apoptosis, 61 synaptic release of neurotransmitters and gene expression (Skalhegg and Tasken, 2000), and a 62 large part of the functional diversity of this kinase results from isoform diversity. Eukaryotic 63 cells express multiple forms of PKA regulatory (R) and catalytic (C) subunits. PKA holoenzyme 64 consists of an R-subunit dimer bound to two C- subunits (R2C2). The biochemical and functional 65 features of PKA holoenzymes are largely determined by the structure and the biochemical 66 properties of the regulatory subunits (Ilouz et al., 2012; Taylor et al., 2012; Zhang et al., 2012). 67 There are two classes of R-subunits, RI and RII, which are classified into α and β 68 subtypes (Doskeland et al., 1993; McKnight et al., 1988). Each isoform is encoded by a unique 69 gene and preferentially expressed in different cells and tissues. RIα and RIIα are ubiquitously 70 expressed in every cell, whereas RIβ and RIIβ expression is more tissue restricted (Cadd and 71 McKnight, 1989). RIβ is expressed in brain and spinal cord (Cadd and McKnight, 1989). RIIβ is 72 predominantly expressed in brain, endocrine, fat, liver and reproductive tissues (Cadd and 3 73 McKnight, 1989; Jahnsen et al., 1986). The four R-subunits are functionally non-redundant. 74 Depletion of either RIβ or RIIβ gene in mice resulted in specific neuronal defects. RIIβ knockout 75 mice display defects in motor behavior and loss of PKA mediated neuronal gene expression 76 (Brandon et al., 1998). Hippocampal slices from RIβ null mice show a severe deficit in long-term 77 depression and depotentiation at the Schaffer collateral-CA1 synapse. Despite a compensatory 78 increase in the RIα protein levels, hippocampal function was not rescued (Brandon et al., 1995). 79 The composition and specific structural and biochemical properties of PKA holoenzymes 80 (R2C2) account, in part, for differential cellular responses to discrete extracellular signals that 81 activate adenylate cyclase (Taylor et al., 2012). Space-restricted kinase activation provides an 82 extra layer of specificity in PKA signaling. PKA is typically targeted at specific intracellular 83 microdomains through interactions with A-Kinase Anchoring Proteins (AKAPs). Many AKAPs 84 have been identified as well as their specific requirements for selective binding to regulatory 85 subunits (Sarma et al., 2010; Wong and Scott, 2004). This spatio-temporal regulation determines 86 the access of proteins to interacting binding partners. AKAPs provide a control mechanism to 87 direct, integrate and locally attenuate the cAMP-initiated cascade. The hallmark signature motif 88 of the AKAPs is an amphipathic helix that binds tightly to the dimerization and docking (D/D) 89 domain of the R-subunits. Recently, a point mutation in the D/D domain of the RIβ gene has 90 been associated with a new neurodegenerative disease presenting with dementia and 91 Parkinsonism, characterized by specific and abundant accumulation of RIβ in neuronal 92 inclusions (Wong et al., 2014). 93 Rigorous cellular characterization of protein localization is a necessary step if we aim to 94 understand PKA function in a physiological context. To date, relatively few efforts have 95 attempted to systematically define the subcellular localization of endogenous proteins using 4 96 imaging-based techniques. Currently, RIβ, the isoform that has a unique role in synaptic 97 plasticity (Brandon et al., 1995) and has been associated with a neurodegenerative disease, is the 98 least studied PKA isoform. RIβ spatial localization has not been systematically studied due to 99 antibody cross reactivity with RIα. Most of the available RIIβ localization data is focused, 100 furthermore, on specific regions of interest; thus the global context of the protein localization is 101 lost. 102 Subcellular cAMP signaling domains are defined by the distinct environments within 103 cellular organelles. The dogma of cAMP-PKA signaling in the nucleus states that, upon cAMP- 104 induced activation of the cytosolic PKA holoenzyme, the C-subunit dissociates from the R- 105 subunit in an isoform-specific manner and translocates into the nucleus via diffusion 106 (Harootunian et al., 1993). Contradictory, reports have increasingly proposed the existence of 107 resident pools of nuclear PKA holoenzyme (Jarnaess et al., 2009; Sample et al., 2012; Zippin et 108 al., 2004). While the necessity of a proper nuclear PKA activity for neuronal function is well- 109 documented the existence of PKA R-subunits or their physiological role within the nucleus has 110 not been well-studied. 111 In this study, we generated high-resolution large-scale mosaic images of several mouse 112 brain slices using RIβ and RIIβ-specific antibodies. Since RIβ is the least studied isoform at the 113 protein level, we focused our analyses on brain regions where we expected RIβ to be 114 predominant based on its mRNA expression profiles and its predicted functional importance 115 from RIβ(-/-) mice. The use of large-scale immunohistochemical brain maps allows us to gain an 116 overview of the RIβ and RIIβ protein distribution over large areas and then zoom in to obtain 117 higher resolution views in order to investigate subcellular features. We found that each 118 regulatory isoform is predominant in distinct brain regions and were able to identify unique and 5 119 consistent patterns of distribution within the hippocampus and the cerebellum. RIβ is 120 concentrated in dendrites, and co-localizes with MAP2, whereas RIIβ is concentrated in axons. 121 We confirmed the RIβ subcellular distribution that emerged from the mosaic images using the 122 mini-Singlet Oxygen Generator (miniSOG) labeling, a probe that allowed us to do correlated 123 light and electron microscopy. We found RIβ at the mitochondria, as we predicted earlier, as well 124 as the nucleus, establishing a new paradigm for PKA signaling in the nucleus. To demonstrate a 125 functional distinction between the two isoforms in the nucleus we selectively down regulated the 126 two isoforms and used CREB phosphorylation as a reporter for a nuclear PKA substrate. Down- 127 regulation of RIβ, but not RIIβ, decreased pCREB in hippocampal cultures. These 128 comprehensive brain images are accessible via Cell Centered Database (CCDB) to browse or 129 download. 130 131 132 133 134 135 136 137 138 139 140 141 6 142 RESULTS 143 Overview of the regional distribution of RIβ and RIIβ across brain regions 144 The overall patterns of RIβ and RIIβ protein distribution across brain regions within a full 145 coronal slice are shown in Figure 1. Images were acquired using confocal microscopy. Image 146 stacks were then knit together to create high-resolution 2D large-scale brain images. The 147 resulting image mosaics provide detailed views of cellular and subcellular distribution of RIβ 148 and RIIβ without losing the context of the tissue.