Elsevier

Experimental Cell Research

Volume 313, Issue 13, 1 August 2007, Pages 2795-2809
Experimental Cell Research

Research Article
Involvement of the catalytic subunit of protein kinase A and of HA95 in pre-mRNA splicing

https://doi.org/10.1016/j.yexcr.2007.05.014Get rights and content

Abstract

Protein kinase A (PKA) is a holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer. Stimulation by cAMP dissociates the holoenzyme and causes translocation to the nucleus of a fraction of the C subunit. Apart from transcription regulation, little is known about the function of the C subunit in the nucleus. In the present report, we show that both Cα and Cβ are localized to spots in the mammalian nucleus. Double immunofluorescence analysis of splicing factor SC35 with the C subunit indicated that these spots are splicing factor compartments (SFCs). Using the E1A in vivo splicing assay, we found that catalytically active C subunits regulate alternative splicing and phosphorylate several members of the SR-protein family of splicing factors in vitro. Furthermore, nuclear C subunits co-localize with the C subunit-binding protein homologous to AKAP95, HA95. HA95 also regulates E1A alternative splicing in vivo, apparently through its N-terminal domain. Localization of the C subunit to SFCs and the E1A splicing pattern were unaffected by cAMP stimulation. Our findings demonstrate that the nuclear PKA C subunit co-locates with HA95 in SFCs and regulates pre-mRNA splicing, possibly through a cAMP-independent mechanism.

Introduction

Protein kinase A (PKA) is considered the main target for cAMP in the cell and regulates a vast number of cellular processes such as metabolism, gene expression, cell growth and differentiation [1]. In the absence of cAMP, PKA is an inactive tetrameric holoenzyme consisting of two catalytic (C) subunits bound to a regulatory (R) subunit dimer, which is compartmentalized to distinct locations in the cell by A-kinase-anchoring proteins (AKAPs) [2]. AKAPs contribute to maintaining specificity of PKA-signaling, but it is also believed that several isoforms and splice variants of both R and C subunits are important mediators of specificity. In mammals, there are four genes encoding R-subunits, RIα, RIβ, RIIα and RIIβ, and four genes encoding C subunits, Cα, Cβ, Cγ and PrKX [1].

When four cAMP molecules bind the R subunits, the holoenzyme dissociates and the C subunits are released to phosphorylate a diverse number of cytosolic target substrates in the vicinity of the PKA holoenzyme. However, upon activation, a proportion of the C subunit translocates to the nucleus [3], [4]. The major targets for C subunit phosphorylation in the nucleus are a group of cAMP-responsive nuclear factors, which bind and regulate the expression of genes containing cAMP-responsive elements (CREs), called CRE-binding proteins (CREBs) [5]. The understanding of how specificity of the nuclear C is maintained is sparse; however, it is expected that temporal and spatial regulation may involve C subunit targeting to nuclear structures independently of the R subunit. This is supported by recent reports demonstrating that the C subunit associates with the nuclear proteins AKIP (A-kinase interacting protein) [6] and HA95 [7].

In this paper, we show that both Cα and Cβ are localized to splicing factor compartments (SFCs), where they co-localize with the C subunit-binding protein HA95, and not AKIP. Furthermore, we demonstrate that the C subunits, as well as HA95, are involved in pre-mRNA splicing, possibly through a cAMP-independent mechanism.

Section snippets

Expression plasmids

Plasmid encoding full-length native Cα1 in the mammalian expression vector pEF-DEST 51 has been described previously [8]. This plasmid was also used for generation of a catalytically inactive mutant by altering lysine 73 to a methionine (Cα1K73M) [9]. This was done using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA, USA) according to manufacturer's protocol using the following primers: Sense; 5′GAACCACTATGCCATGATGATCCTCGACAAACAGA and Antisense; 5′TCTGTTTGTCGAGGATCATCATGGCATAGTGGTTC

PKA C subunits are localized in nuclear spots

A fraction of the PKA C subunit, when not bound to the R subunit, is located in the nucleus of eukaryotic cells [3], [4]. However, few reports demonstrate specific targeting of the C subunit within the nucleus. Immunofluoresence (IF) analysis of the teratocarcinoma cell line NT2 using a pan-C antibody (anti-Cα, see Materials and methods) revealed a diffuse staining in the cytoplasm as well as the nucleus after paraformaldehyde (PF) fixation (Fig. 1A, upper panel). Additionally, a distinct

Discussion

In the present report we show that the C subunit is localized to SFCs in the nucleus. We also demonstrate that catalytically active C subunits regulate alternative splice site selection in vivo. Furthermore, the C subunit-binding protein HA95 is also localized to SFCs and affects the E1A splicing pattern. Interestingly, neither SFC localization of C nor the E1A splicing pattern was affected by cAMP stimulation. This may imply that cAMP-independent mechanisms are involved in PKA-mediated

Acknowledgments

We would like to thank Dr. Adrian Krainer, Cold Spring Harbor Laboratory, NY, USA for providing plasmid encoding the E1A reporter gene (pMTE1A), Dr. Susan Taylor, Department of Chemistry and Biochemistry, Howard Hughes Medical Institute, University of California, San Diego, USA for providing rabbit polyclonal anti-AKIP1, Dr. Gerald Thiel for providing plasmid encoding FLAG®-tagged Cα1 containing an NLS signal and Tove Myhre, Department of Pediatric Research, University of Oslo, Norway for

References (41)

  • C. Westberg et al.

    A novel shuttle protein binds to RNA helicase A and activates the retroviral constitutive transport element

    J. Biol. Chem.

    (2000)
  • J.P. Yang et al.

    Mapping the functional domains of HAP95, a protein that binds RNA helicase A and activates the constitutive transport element of type D retroviruses

    J. Biol. Chem.

    (2001)
  • B.S. Skalhegg et al.

    Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA

    Front. Biosci.

    (2000)
  • W. Wong et al.

    AKAP signalling complexes: focal points in space and time

    Nat. Rev., Mol. Cell Biol.

    (2004)
  • E.A. Nigg et al.

    Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the Golgi complex to the nucleus

    EMBO J.

    (1985)
  • A.T. Harootunian et al.

    Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion

    Mol. Biol. Cell

    (1993)
  • M. Montminy

    Transcriptional regulation by cyclic AMP

    Annu. Rev. Biochem.

    (1997)
  • M. Sastri et al.

    A-kinase-interacting protein localizes protein kinase A in the nucleus

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • I. Han et al.

    Protein kinase A associates with HA95 and affects transcriptional coactivation by Epstein–Barr virus nuclear proteins

    Mol. Cell. Biol.

    (2002)
  • S. Orstavik et al.

    Identification and characterization of novel PKA holoenzymes in human T lymphocytes

    FEBS J.

    (2005)
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