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Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

EFFECT ON PKA LOCALIZATION AND P-Rex1 SIGNALING*
  • Author Footnotes
    1 Both authors contributed equally to this work.
    ,
    Author Footnotes
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Lydia Chávez-Vargas
    Footnotes
    1 Both authors contributed equally to this work.
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Affiliations
    From the Departments of Pharmacology and
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    ,
    Author Footnotes
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Sendi Rafael Adame-García
    Footnotes
    1 Both authors contributed equally to this work.
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Affiliations
    Cell Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, 07360 Mexico,
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  • Author Footnotes
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Rodolfo Daniel Cervantes-Villagrana
    Footnotes
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Affiliations
    From the Departments of Pharmacology and
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  • Author Footnotes
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Alejandro Castillo-Kauil
    Footnotes
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
    Affiliations
    Cell Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, 07360 Mexico,
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  • Jessica G.H. Bruystens
    Affiliations
    Departments of Chemistry and Biochemistry and
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  • Shigetomo Fukuhara
    Affiliations
    Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute (NCVC), Osaka, 565-8565 Japan, and
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  • Susan S. Taylor
    Affiliations
    Departments of Chemistry and Biochemistry and

    Pharmacology, University of California San Diego, La Jolla, California 92093
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  • Naoki Mochizuki
    Affiliations
    Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute (NCVC), Osaka, 565-8565 Japan, and
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  • Guadalupe Reyes-Cruz
    Affiliations
    Cell Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, 07360 Mexico,
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  • José Vázquez-Prado
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology, CINVESTAV-IPN. Av. Instituto Politécnico Nacional 2508. Col. San Pedro Zacatenco, 07360 México, D.F., Mexico. Tel.: 52–55-5747-3380; Fax: 52-55-5747-3394; E-mail: .
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    From the Departments of Pharmacology and
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  • Author Footnotes
    * This work was supported by CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico) Grants 152434 (to J. V.-P.) and 240119 (to G. R.-C.).
    1 Both authors contributed equally to this work.
    2 Supported by fellowships from CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico).
Open AccessPublished:January 21, 2016DOI:https://doi.org/10.1074/jbc.M115.712216
      Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA.

      Introduction

      Rho guanine exchange factors (RhoGEFs)
      The abbreviations used are: RhoGEF, Rho guanine exchange factor; GEF, guanine nucleotide exchange factor; P-Rex1, phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein; PRKAR1a, cAMP-dependent protein kinase type I-α regulatory subunit; PRKACa, cAMP-dependent protein kinase catalytic subunit α; AKAP, A-kinase anchoring protein; PKAS, PKA substrate; (p)CREB, (phospho)-cAMP response element binding; S1P, sphingosine 1-phosphate; DEP, Dishevelled, Egl-10, and Pleckstrin; PDZ domains, mTOR, mammalian target of rapamycin; SDF-1, stromal cell-derived factor 1; HUVEC, human umbilical vein endothelial cell; PAE, porcine aortic endothelial; DH, homology domain; PH, pleckstrin homology; IBMX, isobutylmethylxanthine; EGFP, enhanced GFP; CNB B, cyclic nucleotide-binding domain B.
      are mechanistically linked to fundamental cellular processes, such as migration, adhesion, and morphogenesis. Based on their ability to integrate signaling inputs that result in the activation of Rho GTPases, RhoGEFs indirectly contribute to establish nucleation sites for actin polymerization, thus exerting a tight control on cytoskeleton dynamics (
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      ). This RacGEF is activated by Gβγ and phosphatidylinositol-3,4,5-trisphosphate (
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      ) as well as by direct interaction with mTORC2, a fundamental multimeric kinase with affinity for P-Rex1 DEP domains (
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      ) and by protein phosphatase 1α, which dephosphorylates P-Rex1 at Ser-1165 (
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      ). In endothelial cells, in which this guanine nucleotide exchange factor (GEF) is among the most highly expressed RhoGEFs (
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      ), it influences changes in cell morphology in response to PDGF (
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      P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.
      ,
      • Barber M.A.
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      • Thelen M.
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      The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α.
      ) and participates in the chemotactic response to sphingosine 1-phosphate and SDF-1 (
      • Ledezma-Sánchez B.A.
      • García-Regalado A.
      • Guzmán-Hernández M.L.
      • Vázquez-Prado J.
      Sphingosine-1-phosphate receptor S1P1 is regulated by direct interactions with P-Rex1, a Rac guanine nucleotide exchange factor.
      ,
      • Carretero-Ortega J.
      • Walsh C.T.
      • Hernández-García R.
      • Reyes-Cruz G.
      • Brown J.H.
      • Vázquez-Prado J.
      Phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex-1), a guanine nucleotide exchange factor for Rac, mediates angiogenic responses to stromal cell-derived factor-1/chemokine stromal cell derived factor-1 (SDF-1/CXCL-12) linked to Rac activation, endothelial cell migration, and in vitro angiogenesis.
      ). P-Rex1 structure is constituted by a DH-PH cassette characteristic of the family of RhoGEFs with homology to Dbl (homology (DH) domain and a pleckstrin homology (PH) domain)followed by two DEP domains in tandem, two PDZ domains, and a long C terminus with homology to inositol polyphosphate-4-phosphatase (
      • Welch H.C.
      • Coadwell W.J.
      • Ellson C.D.
      • Ferguson G.J.
      • Andrews S.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Hawkins P.T.
      • Stephens L.R.
      P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.
      ). Phosphorylation of P-Rex1 exerts a positive or negative role on its activity, putatively depending on the kinase involved and the phosphorylation site (
      • Barber M.A.
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      • Beullens M.
      • Ceulemans H.
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      ,
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      ). PKA, in particular, phosphorylates and prevents P-Rex1 from being activated by Gβγ and phosphatidylinositol-3,4,5-trisphosphate (
      • Mayeenuddin L.H.
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      Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gβγ-mediated regulation of its activity.
      ,
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      Domain-domain interaction of P-Rex1 is essential for the activation and inhibition by G protein βγ subunits and PKA.
      ). Although the precise mechanisms of this regulation remain unknown, these findings open the interesting possibility that PKA might maintain interactions with P-Rex1 and also might be regulated by this RacGEF. Here we identified type I PKA as a novel P-Rex1 interactor and study the molecular aspects and functional consequences of this interaction on P-Rex1 regulation and PKA localization.

      Discussion

      P-Rex1 is a multidomain GEF that couples G-protein-coupled receptor signaling to Rac activation through its binding to Gβγ and phosphatidylinositol-3,4,5-trisphosphate (
      • Welch H.C.
      • Coadwell W.J.
      • Ellson C.D.
      • Ferguson G.J.
      • Andrews S.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Hawkins P.T.
      • Stephens L.R.
      P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.
      ,
      • Welch H.C.
      Regulation and function of P-Rex family Rac-GEFs.
      ). In the current study we demonstrate that the type I PKA regulatory subunit α (PRKAR1a) interacts with P-Rex1 and translocates with it to the cell membrane upon SDF-1 stimulation. Moreover, we demonstrate that PKA phosphorylates P-Rex1 at Ser-436 promoting intramolecular inhibitory interactions. Thus we reveal that PKA and P-Rex1 are reciprocally regulated. We previously showed that interaction of the oligomeric kinase mTORC2 with P-Rex1 DEP domains leads to Rac activation and cell migration (
      • Hernández-Negrete I.
      • Carretero-Ortega J.
      • Rosenfeldt H.
      • Hernández-García R.
      • Calderón-Salinas J.V.
      • Reyes-Cruz G.
      • Gutkind J.S.
      • Vázquez-Prado J.
      P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration.
      ). Indeed, mTOR was the first kinase identified as a direct interactor of P-Rex1 (
      • Hernández-Negrete I.
      • Carretero-Ortega J.
      • Rosenfeldt H.
      • Hernández-García R.
      • Calderón-Salinas J.V.
      • Reyes-Cruz G.
      • Gutkind J.S.
      • Vázquez-Prado J.
      P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration.
      ). Subsequent studies demonstrate that AKT1 interacts with P-Rex1 and phosphorylation of this kinase by mTORC2 is facilitated by its interaction with P-Rex1 (
      • Kim E.K.
      • Yun S.J.
      • Ha J.M.
      • Kim Y.W.
      • Jin I.H.
      • Yun J.
      • Shin H.K.
      • Song S.H.
      • Kim J.H.
      • Lee J.S.
      • Kim C.D.
      • Bae S.S.
      Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis.
      ). Thus, in addition to the role attributed to P-Rex1 as an integrator of G-protein-coupled receptor and growth factor receptor signaling leading to Rac activation and cell migration, we show here that this RacGEF is mechanistically regulated by PKA via a multistep mechanism involving direct interactions and phosphorylation-dependent intramolecular inhibitory interactions that interfere with the catalytic DH-PH cassette of P-Rex1, whereas activation of this RacGEF brings PKA to the plasma membrane, revealing a novel scenario of reciprocal regulation between these important signaling proteins.
      We also show here that PKARIa is recruited to P-Rex1 by a non-cannonical mechanism that involves interaction with the P-Rex1 PDZ domains with the CNB-B domain of PKARIa. Although the C terminus of PRKAR1A contains a class II PDZ motif that we initially considered as the canonical motif for interaction with P-Rex1 PDZ domains, arrays using various peptides representing different regions of R1α revealed that CNB B, the second cAMP binding domain of R1α, preferentially interacts with P-Rex1-PDZ1. This interaction was effective even in the absence of the last three amino acids of R1α. There is a clear contrast between this novel interaction described for P-Rex1 and PKA and the classical mechanism of interaction of PKA with AKAPs, which regularly occurs through the N terminus of types I and II regulatory subunits (
      • Huang L.J.
      • Durick K.
      • Weiner J.A.
      • Chun J.
      • Taylor S.S.
      Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits.
      ). Detailed biochemical, cellular, and structural studies have revealed a plethora of AKAPs that interact with the N terminus of PKA regulatory subunits and provide a mechanistic basis by which type II holoenzymes, in particular, are localized to different subcellular compartments (
      • Lissandron V.
      • Zaccolo M.
      Compartmentalized cAMP/PKA signalling regulates cardiac excitation-contraction coupling.
      ,
      • Johnson K.R.
      • Nicodemus-Johnson J.
      • Carnegie G.K.
      • Danziger R.S.
      Molecular evolution of A-kinase anchoring protein (AKAP)-7: implications in comparative PKA compartmentalization.
      ,
      • Scott J.D.
      • Dessauer C.W.
      • Taskén K.
      Creating order from chaos: cellular regulation by kinase anchoring.
      ,
      • Dessauer C.W.
      Adenylyl cyclase: A-kinase anchoring protein complexes: the next dimension in cAMP signaling.
      ). Less information is available for type I PKA, although some AKAPs are dual specific and bind to both RI and RII subunits, whereas a few such as sphingosine kinase-interacting protein (SKIP) and the recently discovered small myristoylated and palmitoylated AKAP are RI-specific (
      • Means C.K.
      • Lygren B.
      • Langeberg L.K.
      • Jain A.
      • Dixon R.E.
      • Vega A.L.
      • Gold M.G.
      • Petrosyan S.
      • Taylor S.S.
      • Murphy A.N.
      • Ha T.
      • Santana L.F.
      • Tasken K.
      • Scott J.D.
      An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria.
      ,
      • Kovanich D.
      • van der Heyden M.A.
      • Aye T.T.
      • van Veen T.A.
      • Heck A.J.
      • Scholten A.
      Sphingosine kinase interacting protein is an A-kinase anchoring protein specific for type I cAMP-dependent protein kinase.
      • Burgers P.P.
      • Ma Y.
      • Margarucci L.
      • Mackey M.
      • van der Heyden M.A.
      • Ellisman M.
      • Scholten A.
      • Taylor S.S.
      • Heck A.J.
      A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane.
      ). Sphingosine kinase interacting protein (SKIP) was also reported to be an R1-specific AKAP (
      • Means C.K.
      • Lygren B.
      • Langeberg L.K.
      • Jain A.
      • Dixon R.E.
      • Vega A.L.
      • Gold M.G.
      • Petrosyan S.
      • Taylor S.S.
      • Murphy A.N.
      • Ha T.
      • Santana L.F.
      • Tasken K.
      • Scott J.D.
      An entirely specific type I A-kinase anchoring protein that can sequester two molecules of protein kinase A at mitochondria.
      ,
      • Kovanich D.
      • van der Heyden M.A.
      • Aye T.T.
      • van Veen T.A.
      • Heck A.J.
      • Scholten A.
      Sphingosine kinase interacting protein is an A-kinase anchoring protein specific for type I cAMP-dependent protein kinase.
      ). Another small myristoylated and palmitoylated AKAP was also shown to be RI-specific and is recruited to the plasma membrane (
      • Burgers P.P.
      • Ma Y.
      • Margarucci L.
      • Mackey M.
      • van der Heyden M.A.
      • Ellisman M.
      • Scholten A.
      • Taylor S.S.
      • Heck A.J.
      A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane.
      ). R1α was also shown to be important for cell migration in response to α4 integrin, and recruitment of PKARIa also did not involve a canonical AKAP mechanism (
      • Tkachenko E.
      • Sabouri-Ghomi M.
      • Pertz O.
      • Kim C.
      • Gutierrez E.
      • Machacek M.
      • Groisman A.
      • Danuser G.
      • Ginsberg M.H.
      Protein kinase A governs a RhoA-RhoGDI protrusion-retraction pacemaker in migrating cells.
      ,
      • Lim C.J.
      • Kain K.H.
      • Tkachenko E.
      • Goldfinger L.E.
      • Gutierrez E.
      • Allen M.D.
      • Groisman A.
      • Zhang J.
      • Ginsberg M.H.
      Integrin-mediated protein kinase A activation at the leading edge of migrating cells.
      • Lim C.J.
      • Han J.
      • Yousefi N.
      • Ma Y.
      • Amieux P.S.
      • McKnight G.S.
      • Taylor S.S.
      • Ginsberg M.H.
      α4 integrins are type I cAMP-dependent protein kinase-anchoring proteins.
      ). The non-canonical interaction that type I PKA establishes with P-Rex1 is the first in which CNB B, the second cAMP binding domain of this regulatory subunit, is recognized as the interacting interface with a protein other than the catalytic subunit of the kinase. Whether this represents the initial example of a common mechanism by which PDZ-containing proteins might control type I PKA subcellular dynamics and localization, equivalent to the role played by AKAPs for type II PKA, or might lead to the identification of additional cAMP-dependent R1α-regulated targets emerge as interesting possibilities that will be the focus of future studies. Interestingly, cAMP-bound RII binds to Gαi and sensitizes δ-opioid Gi-coupled receptors signaling to ERK activation (
      • Stefan E.
      • Malleshaiah M.K.
      • Breton B.
      • Ear P.H.
      • Bachmann V.
      • Beyermann M.
      • Bouvier M.
      • Michnick S.W.
      PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades.
      ).
      Understanding the particular intricacies by which PKA regulates Rho GTPases and cytoskeleton dynamics depends on the identification of specific targets and interacting partners that provide specificity for the effects of this serine/threonine kinase. These elements would help to explain the apparently conflicting results showing that PKA either promotes (
      • Feng H.
      • Hu B.
      • Vuori K.
      • Sarkaria J.N.
      • Furnari F.B.
      • Cavenee W.K.
      • Cheng S.Y.
      EGFRvIII stimulates glioma growth and invasion through PKA-dependent serine phosphorylation of Dock180.
      • Takahashi M.
      • Dillon T.J.
      • Liu C.
      • Kariya Y.
      • Wang Z.
      • Stork P.J.
      Protein kinase A-dependent phosphorylation of Rap1 regulates its membrane localization and cell migration.
      ,
      • Di Zazzo E.
      • Feola A.
      • Zuchegna C.
      • Romano A.
      • Donini C.F.
      • Bartollino S.
      • Costagliola C.
      • Frunzio R.
      • Laccetti P.
      • Di Domenico M.
      • Porcellini A.
      The p85 regulatory subunit of PI3K mediates cAMP-PKA and insulin biological effects on MCF-7 cell growth and motility.
      • Zimmerman N.P.
      • Roy I.
      • Hauser A.D.
      • Wilson J.M.
      • Williams C.L.
      • Dwinell M.B.
      Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells.
      ) or interferes (
      • Burdyga A.
      • Conant A.
      • Haynes L.
      • Zhang J.
      • Jalink K.
      • Sutton R.
      • Neoptolemos J.
      • Costello E.
      • Tepikin A.
      cAMP inhibits migration, ruffling, and paxillin accumulation in focal adhesions of pancreatic ductal adenocarcinoma cells: effects of PKA and EPAC.
      • Lu Q.
      • Tong B.
      • Luo Y.
      • Sha L.
      • Chou G.
      • Wang Z.
      • Xia Y.
      • Dai Y.
      Norisoboldine suppresses VEGF-induced endothelial cell migration via the cAMP-PKA-NF-κB/Notch1 pathway.
      ,
      • Mizuno R.
      • Kamioka Y.
      • Kabashima K.
      • Imajo M.
      • Sumiyama K.
      • Nakasho E.
      • Ito T.
      • Hamazaki Y.
      • Okuchi Y.
      • Sakai Y.
      • Kiyokawa E.
      • Matsuda M.
      In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.
      ,
      • Lee J.W.
      • Lee J.
      • Moon E.Y.
      HeLa human cervical cancer cell migration is inhibited by treatment with dibutyryl-cAMP.
      • O'Leary A.P.
      • Fox J.M.
      • Pullar C.E.
      β-Adrenoceptor activation reduces both dermal microvascular endothelial cell migration via a cAMP-dependent mechanism and wound angiogenesis.
      ,
      • Welch H.C.
      • Coadwell W.J.
      • Ellson C.D.
      • Ferguson G.J.
      • Andrews S.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Hawkins P.T.
      • Stephens L.R.
      P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.
      ) with cell migration. Clearly such regulation is likely to be cell-specific. Although the PKA consensus phosphorylation sequence is present in thousands of potential protein substrates, its exquisite specificity is in part achieved through tight control of its subcellular distribution, and this is mediated in large part by its regulatory subunits that interact with a variety of targeting proteins. Here we identify a new class of PKA targeting protein that functions not only to target but also to bring a set of signaling domains to the membrane in response to G-protein-coupled receptor activation. Furthermore, we found that interaction of type I PKA with P-Rex1 PDZ-domains facilitates the phosphorylation of this GEF and its desensitization. Whether this interaction also facilitates the ability of type I PKA to recognize other substrates emerges as an interesting open question. Considering the mobilization of type I PKA to the cell membrane together with P-Rex1, upon stimulation of cells with SDF-1, it will be interesting to explore whether this interaction facilitates the potential regulation by PKA of plasma membrane proteins involved in chemotactic responses. Thus, it is likely that RI subunits play a far more important role at the plasma membrane than was previously appreciated.
      Bachmann et al. (
      • Bachmann V.A.
      • Riml A.
      • Huber R.G.
      • Baillie G.S.
      • Liedl K.R.
      • Valovka T.
      • Stefan E.
      Reciprocal regulation of PKA and Rac signaling.
      ) recently described the formation of a transient complex between Rac1 and PKA through the PKA RIIβ regulatory subunit, and this interaction depends on the AKAP-like behavior of Rac1. Such an interaction would provide reciprocal regulation for signaling cascades of both components; particularly, Rac1-GTP stabilizes type II PKA holoenzyme (
      • Bachmann V.A.
      • Riml A.
      • Huber R.G.
      • Baillie G.S.
      • Liedl K.R.
      • Valovka T.
      • Stefan E.
      Reciprocal regulation of PKA and Rac signaling.
      ). Here, we describe a new function for P-Rex1 interacting with PKA holoenzyme through the regulatory subunit Iα. This represents an alternative way of assembling a PKA signaling complex that is different from the traditional AKAP-mediated mechanism.
      Our results confirm the previously reported ability of PKA to inhibit P-Rex1 signaling (
      • Mayeenuddin L.H.
      • Garrison J.C.
      Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gβγ-mediated regulation of its activity.
      ,
      • Urano D.
      • Nakata A.
      • Mizuno N.
      • Tago K.
      • Itoh H.
      Domain-domain interaction of P-Rex1 is essential for the activation and inhibition by G protein βγ subunits and PKA.
      ). Furthermore, we extend these findings revealing a specific interaction of type I PKA CNB B preferentially with P-Rex1 PDZ1, the precise identification of the phosphorylation site located at Ser-436 at the first DEP domain of this GEF, and the mechanism of inhibition regulated by intramolecular interactions promoted by phosphorylation. Based on these findings and the influence of P-Rex1 on the localization of PKA, we propose a working model of P-Rex1 regulation by PKA (Fig. 8). Accordingly, P-Rex1 carries type I PKA as its own regulator. Thus, during a chemotactic response, the activity of P-Rex1 is fine-tuned by PKA, which accompanies the GEF when it goes to the membrane. Mechanistically, PKA attenuates P-Rex1 activity via two alternative inhibitory intramolecular interactions acting directly on the DH-PH module; one involving the phosphorylation of Ser-436 at DEP1 domain and the second the C-terminal region in which the action of PKA is likely indirect. According to our model, PKA switches the interactions between P-Rex1 N- and C-terminal regions, needed to keep the GEF sensitive to its activators (
      • Barber M.A.
      • Hendrickx A.
      • Beullens M.
      • Ceulemans H.
      • Oxley D.
      • Thelen S.
      • Thelen M.
      • Bollen M.
      • Welch H.C.
      The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α.
      ,
      • Urano D.
      • Nakata A.
      • Mizuno N.
      • Tago K.
      • Itoh H.
      Domain-domain interaction of P-Rex1 is essential for the activation and inhibition by G protein βγ subunits and PKA.
      ) toward inhibitory interactions within the N-terminal domains that depend on the phosphorylation of Ser-436. In summary, evidence shown here contributes to understanding how temporal regulation of P-Rex1 by PKA occurs. The fact that P-Rex1 is physically associated with PKA could partly explain how this GEF is tightly controlled during a chemotactic event, contributing to define a mechanism by which spatial organization of chemotactic signaling is achieved.
      Figure thumbnail gr8
      FIGURE 8Working model to explain the reciprocal regulation between PKA and P-Rex1 during a chemotactic response. P-Rex1 is an effector of Gβγ and phosphatidylinositol-3,4,5-trisphosphate (PIP3)-activated downstream of chemotactic G protein-coupled receptors (
      • Carretero-Ortega J.
      • Walsh C.T.
      • Hernández-García R.
      • Reyes-Cruz G.
      • Brown J.H.
      • Vázquez-Prado J.
      Phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex-1), a guanine nucleotide exchange factor for Rac, mediates angiogenic responses to stromal cell-derived factor-1/chemokine stromal cell derived factor-1 (SDF-1/CXCL-12) linked to Rac activation, endothelial cell migration, and in vitro angiogenesis.
      ,
      • Welch H.C.
      • Coadwell W.J.
      • Ellson C.D.
      • Ferguson G.J.
      • Andrews S.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Hawkins P.T.
      • Stephens L.R.
      P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.
      ,
      • Welch H.C.
      Regulation and function of P-Rex family Rac-GEFs.
      ). P-Rex1 activates Rac, and it is inactivated by PKA (
      • Mayeenuddin L.H.
      • Garrison J.C.
      Phosphorylation of P-Rex1 by the cyclic AMP-dependent protein kinase inhibits the phosphatidylinositiol (3,4,5)-trisphosphate and Gβγ-mediated regulation of its activity.
      ). In response to agonists such as SDF-1, P-Rex1 translocates to the plasma membrane to where it carries type I PKA, which interacts with P-Rex1 PDZ domains (preferentially PDZ1) via the CNB B domain of its regulatory subunit. Mechanistically, PKA promotes intramolecular inhibitory interactions within the N-terminal region occurring between DEP1, phosphorylated by PKA at Ser-476, and the DH-PH module. In addition, PKA enhances the inhibitory potential of P-Rex1-C-terminal region, putatively via an unidentified PKA-regulated kinase. Phosphorylated P-Rex1 is kept in this inactive conformation that maintains the DH-PH module inaccessible to Rac. Dephosphorylation of P-Rex1 at Ser(P)-1165 by protein phosphatase 1α has been reported as a mechanism for activation of this RacGEF (
      • Barber M.A.
      • Hendrickx A.
      • Beullens M.
      • Ceulemans H.
      • Oxley D.
      • Thelen S.
      • Thelen M.
      • Bollen M.
      • Welch H.C.
      The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α.
      ). Additional inhibitory and stimulatory phosphorylation sites on P-Rex1 by unidentified kinases have been described (
      • Montero J.C.
      • Seoane S.
      • Ocaña A.
      • Pandiella A.
      P-Rex1 participates in Neuregulin-ErbB signal transduction and its expression correlates with patient outcome in breast cancer.
      ). PIP2, phosphatidylinositol-4,5-bisphosphate.

      Author Contributions

      L. C.-V. performed the two-hybrid screening and designed, performed, and analyzed most of the experiments in Figs. 1, 3C, 6G, and 7E. S. R. A.-G. designed, performed, and analyzed the experiments shown in Figs. 1, F and I, 3B (some repetitions), 4, 5C, and most of the experiments in FIGURE 6, FIGURE 7, B, C, and F. R. D. C.-V. designed, performed, and analyzed the experiments shown in Figs. 1J, 2A, 3A, 3B (some repetitions), and 5A. A. C.-K. designed, performed, and analyzed the experiments shown in Figs. 3D, 5B, and 5D. J. G. H. B. designed, performed, and analyzed the experiments shown in Fig 2B. S. F. and N. M. provided technical assistance and contributed with the overall design, execution, and analysis of the experiments shown in Fig. 3C and contributed to the analysis and interpretation of data. S. S. T. contributed with the overall design and analysis of the experiments shown in Fig. 2B and contributed to the analysis and interpretation of data and edited the final version of the manuscript. G. R.-C. provided technical assistance and contributed to design, analysis, and interpretation of data. J. V. P. conceived and coordinated the study and wrote the paper together with L. C.-V. and S. R. A.-G. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      Technical assistance provided by Estanislao Escobar Islas, Margarita Valadez, David Pérez, and Jaime Estrada Trejo is acknowledged.

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