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• W2032020222 abstract "Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Our results identify a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that the tyrosine phosphatase, Shp2, is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits its protein-tyrosine phosphatase activity. Given the important cardiac roles of both AKAP-Lbc and Shp2, we investigated the AKAP-Lbc-Shp2 interaction in the heart. AKAP-Lbc-tethered PKA is implicated in cardiac hypertrophic signaling; however, mechanism of PKA action is unknown. Mutations resulting in loss of Shp2 catalytic activity are also associated with cardiac hypertrophy and congenital heart defects. Our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote compensatory cardiac hypertrophy. Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Our results identify a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that the tyrosine phosphatase, Shp2, is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits its protein-tyrosine phosphatase activity. Given the important cardiac roles of both AKAP-Lbc and Shp2, we investigated the AKAP-Lbc-Shp2 interaction in the heart. AKAP-Lbc-tethered PKA is implicated in cardiac hypertrophic signaling; however, mechanism of PKA action is unknown. Mutations resulting in loss of Shp2 catalytic activity are also associated with cardiac hypertrophy and congenital heart defects. Our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote compensatory cardiac hypertrophy. Organization of the bewildering array of cell signaling proteins into coherent networks is facilitated by scaffold proteins (1Shi F. Lemmon M.A. Biochemistry: KSR plays CRAF-ty.Science. 2011; 332: 1043-1044Crossref PubMed Scopus (5) Google Scholar). A-kinase-anchoring proteins (AKAPs) 3The abbreviations used are: AKAPA-kinase-anchoring proteinBiFCbimolecular fluorescence complementationCFPcyan fluorescent proteinDMSOdimethyl sulfoxideIBMXisobutylmethylxanthineIPimmunoprecipitationPTPprotein-tyrosine phosphataseShp2Src homology domain-containing phosphatase 2VCC terminus of VenusVNN terminus of Venus. are a diverse family of scaffold proteins that form multiprotein complexes functioning to integrate cAMP signaling with other pathways (2Carnegie G.K. Means C.K. Scott J.D. A-kinase anchoring proteins: from protein complexes to physiology and disease.IUBMB Life. 2009; 61: 394-406Crossref PubMed Scopus (140) Google Scholar, 3Wong W. Scott J.D. AKAP signalling complexes: focal points in space and time.Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (850) Google Scholar, 4Beene D.L. Scott J.D. A-kinase anchoring proteins take shape.Curr. Opin. Cell Biol. 2007; 19: 192-198Crossref PubMed Scopus (197) Google Scholar). All members of the AKAP family possess a conserved PKA-anchoring domain as well as binding sites for other signaling components (5Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes.EMBO J. 2001; 20: 1651-1662Crossref PubMed Scopus (180) Google Scholar, 6Pidoux G. Taskén K. Specificity and spatial dynamics of protein kinase A signaling organized by A-kinase-anchoring proteins.J. Mol. Endocrinol. 2010; 44: 271-284Crossref PubMed Scopus (137) Google Scholar). For example, to ensure that signaling is tightly regulated, AKAPs coordinate both signal activators (e.g. protein kinases) and signal terminators (e.g. protein phosphatases) (7Schillace R.V. Scott J.D. Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220.Curr. Biol. 1999; 9: 321-324Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 8Cardone L. Carlucci A. Affaitati A. Livigni A. DeCristofaro T. Garbi C. Varrone S. Ullrich A. Gottesman M.E. Avvedimento E.V. Feliciello A. Mitochondrial AKAP121 binds and targets protein tyrosine phosphatase D1, a novel positive regulator of Src signaling.Mol. Cell. Biol. 2004; 24: 4613-4626Crossref PubMed Scopus (102) Google Scholar, 9Alto N. Carlisle Michel J.J. Dodge K.L. Langeberg L.K. Scott J.D. Intracellular targeting of protein kinases and phosphatases.Diabetes. 2002; 51: S385-S388Crossref PubMed Google Scholar). Here, we report an interaction of the protein-tyrosine phosphatase, Shp2 (PTPN11) with AKAP-Lbc (also termed AKAP13) and demonstrate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart. A-kinase-anchoring protein bimolecular fluorescence complementation cyan fluorescent protein dimethyl sulfoxide isobutylmethylxanthine immunoprecipitation protein-tyrosine phosphatase Src homology domain-containing phosphatase 2 C terminus of Venus N terminus of Venus. Localized regulation and integration of signal transduction are important for proper cardiac function, and perturbation of this leads to heart failure. We are now just beginning to understand the spatiotemporal aspects of AKAP-mediated signaling in the heart. Multiple cardiac AKAPs have been characterized and shown to perform a critical role in mediating the effects of neurohumoral stimulation on the heart by integrating PKA activity with additional enzymes (10Mauban J.R. O'Donnell M. Warrier S. Manni S. Bond M. AKAP-scaffolding proteins and regulation of cardiac physiology.Physiology. 2009; 24: 78-87Crossref PubMed Scopus (61) Google Scholar, 11Diviani D. Dodge-Kafka K.L. Li J. Kapiloff M.S. A-kinase anchoring proteins: scaffolding proteins in the heart.Am. J. Physiol. Heart Circ. Physiol. 2011; 301: H1742-H1753Crossref PubMed Scopus (77) Google Scholar, 12Carnegie G.K. Burmeister B.T. A-kinase anchoring proteins that regulate cardiac remodeling.J. Cardiovasc. Pharmacol. 2011; 58: 451-458Crossref PubMed Scopus (15) Google Scholar). A recent proteomic study suggests that differential expression of AKAPs coupled with alterations in the AKAP “interactome” may be critical factors in heart failure (13Aye T.T. Soni S. van Veen T.A. van der Heyden M.A. Cappadona S. Varro A. de Weger R.A. de Jonge N. Vos M.A. Heck A.J. Scholten A. Reorganized PKA-AKAP associations in the failing human heart.J. Mol. Cell. Cardiol. 2012; 52: 511-518Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Indeed, we have demonstrated previously that AKAP-Lbc expression is up-regulated under hypertrophic conditions in rats as well as in human heart failure samples, promoting cardiac hypertrophy through a protein kinase D1 (PKD1)-mediated mechanism (14Carnegie G.K. Soughayer J. Smith F.D. Pedroja B.S. Zhang F. Diviani D. Bristow M.R. Kunkel M.T. Newton A.C. Langeberg L.K. Scott J.D. AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.Mol. Cell. 2008; 32: 169-179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Cardiac hypertrophy is initially a beneficial, compensatory process, decreasing wall stress and increasing cardiac output and stroke volume. However, prolonged hypertrophy is maladaptive, transitioning to decompensation and cardiac failure. Multiple pathological hypertrophic pathways converge on a set of transcriptional regulators, promoting initiation of a developmental gene-reprogramming paradigm (often termed the fetal gene response). These “fetal” cardiac genes encode proteins involved in contraction, calcium handling, and metabolism, and their activation accompanies cardiac hypertrophy (15Frey N. Olson E.N. Cardiac hypertrophy: the good, the bad, and the ugly.Annu. Rev. Physiol. 2003; 65: 45-79Crossref PubMed Scopus (1188) Google Scholar, 16Heineke J. Molkentin J.D. Regulation of cardiac hypertrophy by intracellular signalling pathways.Nat. Rev. Mol. Cell Biol. 2006; 7: 589-600Crossref PubMed Scopus (1497) Google Scholar). In addition to binding PKD1, AKAP-Lbc acts as Rho-guanine nucleotide exchange factor, implicated in cardiomyocyte hypertrophy (17Appert-Collin A. Cotecchia S. Nenniger-Tosato M. Pedrazzini T. Diviani D. The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates α1-adrenergic receptor-induced cardiomyocyte hypertrophy.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10140-10145Crossref PubMed Scopus (84) Google Scholar), possibly through a p38α mitogen-activated protein kinase (MAPK) pathway (18Cariolato L. Cavin S. Diviani D. A-kinase anchoring protein (AKAP)-Lbc anchors a PKN-based signaling complex involved in α1-adrenergic receptor-induced p38 activation.J. Biol. Chem. 2011; 286: 7925-7937Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). AKAP-Lbc also anchors protein kinase A (PKA) and protein kinase C (PKCα and PKCη isoforms) (19Carnegie G.K. Smith F.D. McConnachie G. Langeberg L.K. Scott J.D. AKAP-Lbc nucleates a protein kinase D activation scaffold.Mol. Cell. 2004; 15: 889-899Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and our previous data suggest a role for PKA in hypertrophic signaling (14Carnegie G.K. Soughayer J. Smith F.D. Pedroja B.S. Zhang F. Diviani D. Bristow M.R. Kunkel M.T. Newton A.C. Langeberg L.K. Scott J.D. AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.Mol. Cell. 2008; 32: 169-179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), but downstream pathways are not clear. Interestingly, Shp2 is also implicated in the modulation of myocyte size, cardiomyopathy, and heart failure (20Nakamura T. Colbert M. Krenz M. Molkentin J.D. Hahn H.S. Dorn 2nd, G.W. Robbins J. Mediating ERK1/2 signaling rescues congenital heart defects in a mouse model of Noonan syndrome.J. Clin. Invest. 2007; 117: 2123-2132Crossref PubMed Scopus (92) Google Scholar, 21Krenz M. Gulick J. Osinska H.E. Colbert M.C. Molkentin J.D. Robbins J. Role of ERK1/2 signaling in congenital valve malformations in Noonan syndrome.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 18930-18935Crossref PubMed Scopus (64) Google Scholar, 22Nakamura T. Gulick J. Colbert M.C. Robbins J. Protein-tyrosine phosphatase activity in the neural crest is essential for normal heart and skull development.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11270-11275Crossref PubMed Scopus (49) Google Scholar). LEOPARD syndrome patients most commonly manifest hypertrophic cardiomyopathy due to mutations in the PTPN11 gene encoding Shp2 that generally results in impaired Shp2 catalytic activity (23Kontaridis M.I. Swanson K.D. David F.S. Barford D. Neel B.G. PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects.J. Biol. Chem. 2006; 281: 6785-6792Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 24Stewart R.A. Sanda T. Widlund H.R. Zhu S. Swanson K.D. Hurley A.D. Bentires-Alj M. Fisher D.E. Kontaridis M.I. Look A.T. Neel B.G. Phosphatase-dependent and -independent functions of Shp2 in neural crest cells underlie LEOPARD syndrome pathogenesis.Dev. Cell. 2010; 18: 750-762Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Here, we observe similar results, showing diminished Shp2 activity associated with AKAP-Lbc in hypertrophic heart samples induced by chronic isoproterenol treatment to activate PKA. Mechanistically, our data suggest that AKAP-Lbc facilitates the phosphorylation of Shp2 by PKA, acting to inhibit Shp2 PTP activity, which may in turn promote cardiac hypertrophy. Anti-V5-agarose, anti-FLAG-agarose and anti-FLAG antibody were from Sigma. Anti-V5 antibody (mouse, 1:5000) and purified recombinant PKD1 were from Invitrogen. Anti-Shp2 antibody (rabbit, 1:1000) was from Santa Cruz and was also kindly provided by Geng Sheng Feng (University of California San Diego) (rabbit, 1:20,000). Anti-phospho-PKA substrate (RRXS*/T*) antibody (1:1000) was from Cell Signaling Technology. Purified recombinant PKA was from Promega. cDNA for Shp2 expression was kindly provided by Geng-Sheng Feng. Bimolecular fluorescence complementation (BiFC) plasmids were originally from Chang-Deng Hu (Purdue University). Full-length AKAP-Lbc was expressed as a RFP and V5-tagged protein in pcDNA3.1, or as FLAG-AKAP-Lbc in pEGFP-N1. pEGFP-N1-AKAP-Lbc-ΔPKA expresses a mutant version of AKAP-Lbc that cannot bind the PKA-RII regulatory subunit (25Diviani D. Soderling J. Scott J.D. AKAP-Lbc anchors protein kinase A and nucleates Gα12-selective Rho-mediated stress fiber formation.J. Biol. Chem. 2001; 276: 44247-44257Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). GST-AKAP-Lbc fusion proteins were produced in BL21(DE3)pLys as described previously (19Carnegie G.K. Smith F.D. McConnachie G. Langeberg L.K. Scott J.D. AKAP-Lbc nucleates a protein kinase D activation scaffold.Mol. Cell. 2004; 15: 889-899Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). HEK293 cells were transfected and lysed as described previously (19Carnegie G.K. Smith F.D. McConnachie G. Langeberg L.K. Scott J.D. AKAP-Lbc nucleates a protein kinase D activation scaffold.Mol. Cell. 2004; 15: 889-899Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). For phosphorylation experiments, the phosphatase inhibitor microcystin-LR was included (100 nm). Lysates were incubated on ice for 10 min and centrifuged at 20,000 × g for 15 min at 4 °C. Cleared lysates were incubated with antibodies for 1 h at 4 °C with rocking, followed by precipitation of antibody-antigen complexes with protein A/G-agarose. Immunoprecipitates were washed 5 × 1 ml in lysis buffer, eluted in SDS-PAGE sample buffer, and separated by SDS-PAGE. GST pulldowns were performed similarly, except that protein complexes were isolated by incubation with glutathione-Sepharose for 1 h at 4 °C. For endogenous protein co-immunoprecipitations (co-IPs) from heart, frozen mouse hearts were homogenized by Polytron in 20 mm HEPES, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.5%, and protease inhibitors. The heart extract was clarified by centrifugation at 20,000 × g for 20 min and then used for IP. Proteomics and informatics services were provided by the CBC-UIC Research Resources Center Mass spectrometry, Metabolomics and Proteomics Facility, established in part by a grant from The Searle Funds at the Chicago Community Trust to the Chicago Biomedical Consortium. BiFC expression constructs consisting of the N terminus or C terminus of Venus (VN and VC, respectively) were originally provided by Chang-Deng Hu. AKAP-Lbc was cloned into the VN vector with the Venus fragment fused to the N terminus. Shp2 was cloned into the VC vector with the Venus fragment at the C terminus. Cells were transiently transfected and then imaged ∼20 h after transfection so that the proteins were not highly overexpressed. CFP was expressed along with the BiFC constructs, as a marker for transfected cells. Confocal images were acquired using a Carl Zeiss LSM 510 mounted on an Axiovert 100 M microscope. Images were obtained using a 514-nm argon laser for YFP and 458 nm for CFP with a Plan-Apochromat 63×/1.4 oil immersion objective lens. A 531–595-nm wavelength bandpass filter was used for YFP emission, and a 470–500-nm wavelength bandpass filter was used for CFP, with a pinhole of 0.7 Airy units, which provides a z resolution of ∼0.6 μm. Following immunoprecipitation of either AKAP-Lbc or Shp2, immune complexes were washed five times with IP buffer (10 mm sodium phosphate buffer, pH 6.95, 150 mm NaCl, 5 mm EDTA, 5 mm EGTA, 1% Triton X-100) before being resuspended in phosphatase assay buffer (50 mm HEPES, 100 mm NaCl, 5 mm DTT, 2 mm Na2EDTA, 0.01% Brij-35, pH 7.5). The phosphatase assay was carried out in a total reaction volume of 50 μl using 30 μm fluorescein diphosphate as substrate. After a 20-min incubation at 30 °C, supernatant was transferred to a 96-well plate, and phosphatase activity was measured using a PHERAstar FS microplate reader, with excitation at 485 nm and emission at 520 nm. For calibration of PTP activity using this assay, T cell PTP (New England Biolabs) was serially diluted and used for assay as described above. Fluorescence intensity was measured for known amounts of enzyme ranging from 0 to 500 milliunits of specific activity. One unit is defined as the amount of enzyme that hydrolyzes 1 nmol of p-nitrophenyl phosphate (50 mm) in 1 min at 30 °C in a total reaction volume of 50 μl. Immunoprecipitated Shp2 was phosphorylated in vitro in kinase assay buffer (25 mm Tris, pH 7.5, 0.1 mm EGTA, 0.1 mm Na3VO4, 0.03% Brij-35, 10 mm MgAc2, 100 mm ATP, 1 mCi of [γ-32P]ATP) supplemented with bacterially purified recombinant PKA C-subunit (0.2 mg), or recombinant PKD1 (0.2 mg), for 20 min at 30 °C. Reactions were terminated by washing twice with fresh kinase buffer prior to resuspension in Laemmli sample buffer or PTP activity assay. 16-week-old, male FVB mice received continuous subcutaneous administration of isoproterenol (25 μg/g per day for 30 days) or saline control via minipump (Alzet) implantation. Hearts were assessed before and after treatment by M-mode echocardiography using a VisualSonics Vevo 770 ultrasound instrument. Hearts were removed from mice, and a small section of the left ventricle was fixed in paraformaldehyde and embedded in paraffin. Paraffin-embedded sections were stained with hematoxylin and eosin. All data are expressed as means ± S.E. Differences in quantitative variables were examined by one-way analysis of variance (ANOVA) or an unpaired two-tailed t test. A p value < 0.05 was considered significant (*), a p value < 0.01 was considered very significant (**), and a p value < 0.001 was considered extremely significant (***). All analyses were performed using InStat. The AKAP-Lbc signaling complex is composed of multiple protein kinases (18Cariolato L. Cavin S. Diviani D. A-kinase anchoring protein (AKAP)-Lbc anchors a PKN-based signaling complex involved in α1-adrenergic receptor-induced p38 activation.J. Biol. Chem. 2011; 286: 7925-7937Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 26Smith F.D. Langeberg L.K. Cellurale C. Pawson T. Morrison D.K. Davis R.J. Scott J.D. AKAP-Lbc enhances cyclic AMP control of the ERK1/2 cascade.Nat. Cell Biol. 2010; 12: 1242-1249Crossref PubMed Scopus (97) Google Scholar); therefore, we wondered whether AKAP-Lbc may also bind protein phosphatases. To identify novel binding partners, we performed multiple proteomic screens using GST-tagged AKAP-Lbc fragments to purify associated proteins, which were identified by tandem MS. This approach identified the tyrosine phosphatase Shp2 as an AKAP-Lbc associated protein (Fig. 1A). The AKAP-Lbc-Shp2 interaction was validated in HEK293 cells by co-precipitation of endogenous Shp2 with AKAP-Lbc (Fig. 1B). In addition, endogenous Shp2 was co-purified with endogenous AKAP-Lbc from heart extract and vice versa (Fig. 1, C and D). Next, we performed mapping studies to define the Shp2 site of interaction with AKAP-Lbc. Binding of Shp2 to a family of immobilized GST-AKAP-Lbc fragments detected an interaction with a central portion of AKAP-Lbc (residues 1388–1922; Fig. 1E). To visualize the interaction of AKAP-Lbc and Shp2 inside cells, we used BiFC. This technique has several advantages over more traditional immunostaining or fluorescent protein localization. In particular, the BiFC method enables us to specifically visualize the Shp2-AKAP-Lbc interaction at potentially low levels of protein expression, without background (i.e. fluorescence is observed only for Shp2-AKAP-Lbc complex formation) (27Kerppola T.K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.Nat. Protoc. 2006; 1: 1278-1286Crossref PubMed Scopus (380) Google Scholar, 28Shyu Y.J. Suarez C.D. Hu C.D. Visualization of ternary complexes in living cells by using a BiFC-based FRET assay.Nat. Protoc. 2008; 3: 1693-1702Crossref PubMed Scopus (61) Google Scholar). A nonfluorescent fragment (VN) from a split Venus fluorescent protein was fused to AKAP-Lbc (VN-AKAP-Lbc), and a nonfluorescent VC fragment was fused to Shp2 fragment (VC-Shp2). Interaction of AKAP-Lbc and Shp2 brings the two nonfluorescent fragments into close proximity, thereby reconstituting a functional fluorescent protein, resulting in fluorescence. Results in Fig. 2 show that Shp2 specifically interacts with AKAP-Lbc in the cytoplasm of HEK293 cells (Fig. 2B). In Fig. 2C (merged image), yellow indicates overlap of AKAP-Lbc-Shp2 complex BiFC signal (green) with α-actinin (red), suggesting that a portion of the AKAP-Lbc-Shp2 complex has a sarcomeric and sarcolemmal localization in cardiac myocytes. We did not observe significant differences in AKAP-Lbc-Shp2 complex formation and localization by BiFC in myocytes under basal and agonist treatment. No fluorescence was observed when VN-AKAP-Lbc was expressed alone or with a noninteracting control VC-protein (VC-PI3K). Similarly, no fluorescence was observed with expression of VC-Shp2 alone or with a control VN-protein (VN-Rab5). Western blot analysis of protein expression confirmed that all proteins were similarly expressed and were not degraded. Collectively, our biochemical studies and imaging data demonstrate that Shp2 is a component of the AKAP-Lbc signaling complex. Immunoprecipitation of endogenous AKAP-Lbc from mouse heart extract followed by in vitro PTP activity assay demonstrates that tyrosine phosphatase activity co-purifies with AKAP-Lbc (Fig. 3A). Shp2 IPs were used as a positive control in these experiments, and sodium orthovanadate was used to inhibit tyrosine phosphatase activity, demonstrating that we are specifically measuring PTP activity. Additionally, our results show that PTP activity is associated with V5-tagged AKAP-Lbc, expressed in HEK293 cells (Fig. 3B). Using two different antibodies in these experiments (anti-AKAP-Lbc and anti-V5) gives us confidence that the phosphatase activity measured is specifically associated with AKAP-Lbc. Importantly, we also carried out measurements of AKAP-Lbc-associated PTP activity from samples where Shp2 had been previously immunodepleted. Results presented in Fig. 3B show a dramatic reduction of PTP activity associated with AKAP-Lbc, suggesting that we are measuring Shp2 activity in the AKAP-Lbc complex. Western blots indicating corresponding levels of AKAP-Lbc and Shp2 from samples used in these assays are shown in Fig. 3C. Our results demonstrate that Shp2 is a component of the AKAP-Lbc complex; however, the function and regulation of Shp2 in the AKAP-Lbc complex are unclear. To determine whether Shp2 is a substrate for either PKA and/or PKD1, we performed in vitro phosphorylation assays using [γ-32P]MgATP with immunopurified Shp2 and purified recombinant PKA and PKD1 (Fig. 4A). Following SDS-PAGE and transfer to nitrocellulose, autoradiography results demonstrate that Shp2 is phosphorylated by PKA but not by PKD1. PKD1 autophosphorylation as well as a positive control using HDAC5 (a well characterized PKD1 substrate) indicates that the PKD1 was active in this assay (Fig. 4A, top panel). Western blotting for Shp2 confirmed that equivalent levels of Shp2 were present in both the PKA and PKD1 reaction (Fig. 4A, bottom panel). To determine whether PKA phosphorylates Shp2 in vivo, we expressed FLAG-Shp2 in HEK293 cells and then treated these cells with forskolin and IBMX to activate PKA, prior to lysis. Following immunoprecipitation of Shp2, SDS-PAGE and transfer to nitrocellulose, we performed Western blotting using an anti-PKA-phosphosubstrate antibody. This antibody recognizes PKA substrate phosphorylation with the consensus sequence R-R-X-pS/pT. Results in Fig. 4B show that Shp2 phosphorylation is barely detected under basal conditions, whereas Shp2 is phosphorylated in response to PKA activation. Similarly, Shp2 is phosphorylated by PKA in cardiac myocytes in response to isoproterenol stimulation (Fig. 4C). Overall, these results indicate that Shp2 is a PKA substrate. Next, we determined whether PKA activation and subsequent Shp2 phosphorylation modulate Shp2 activity. HEK293 cells were untreated or treated with forskolin/IBMX for 20 min prior to lysis, AKAP-Lbc immunoprecipitation, and PTP assay. Activation of PKA reduces AKAP-Lbc-associated PTP activity, suggesting that PKA inhibits Shp2 activity in the AKAP-Lbc complex (Fig. 5A). The levels of AKAP-Lbc and associated Shp2 in these assays were determined by Western blotting, indicating similar levels of AKAP-Lbc and co-immunoprecipitating Shp2 in all conditions (Fig. 5B). Together, these results indicate that PKA does not affect the association of Shp2 with AKAP-Lbc, but acts to inhibit Shp2 catalytic activity in the AKAP-Lbc complex. For direct evidence that PKA phosphorylation of Shp2 inhibits Shp2 catalytic activity we phosphorylated Shp2 in vitro with purified PKA and then performed a PTP activity assay. Results indicate that Shp2 is phosphorylated (Fig. 5D) and displays reduced PTP activity compared with untreated Shp2 (Fig. 5C). Next, we tested whether AKAP-Lbc facilitates PKA phosphorylation of Shp2 by anchoring PKA. We used a mutant form of AKAP-Lbc that cannot bind PKA, termed AKAP-Lbc-ΔPKA. The AKAP-Lbc-ΔPKA mutant has two amino acid substitutions (A1251P/I1260P) in the PKA-RII binding region that disrupt the amphipathic helix structure required for hydrophobic binding to the regulatory subunit of PKA, thereby disrupting PKA association (29Diviani D. Scott J.D. AKAP signaling complexes at the cytoskeleton.J. Cell Sci. 2001; 114: 1431-1437Crossref PubMed Google Scholar). HEK293 cells expressing wild-type AKAP-Lbc or the AKAP-Lbc-ΔPKA mutant were untreated or treated with forskolin/IBMX for 20 min prior to lysis, AKAP-Lbc immunoprecipitation, and PTP assay. Activation of PKA significantly reduces wild-type AKAP-Lbc (WT-AKAP-Lbc)-associated PTP activity, but not PTP activity associated with the AKAP-Lbc-ΔPKA mutant (Fig. 5E). Western blot analysis demonstrates equal expression of both WT-AKAP-Lbc and AKAP-Lbc-ΔPKA and equal co-immunoprecipitation of Shp2 (Fig. 5F). Using the PKA-phosphosubstrate antibody we examined the extent of Shp2 phosphorylation in Shp2 associated with either WT-AKAP-Lbc or the AKAP-Lbc-ΔPKA mutant. Results show that PKA phosphorylation of Shp2 associated with AKAP-Lbc is barely detectable under basal conditions. In response to forskolin/IBMX treatment, Shp2 phosphorylation is significantly diminished when Shp2 is co-expressed with the AKAP-Lbc-ΔPKA mutant compared with the WT-AKAP-Lbc complex (Fig. 5, F and G). By carrying out an additional experiment, where we precleared AKAP-Lbc (and associated Shp2) from cell lysate prior to Shp2 immunoprecipitation, our results demonstrate that the Shp2 not associated with AKAP-Lbc is minimally phosphorylated by PKA, in contrast to the Shp2 that is associated with AKAP-Lbc (Fig. 5H). Collectively, these data suggest that AKAP-Lbc plays an important role in the regulation of Shp2 activity by facilitating phosphorylation of Shp2 by PKA. In summary, phosphorylation of Shp2 by AKAP-Lbc-anchored PKA inhibits Shp2 activity and does not affect the association of Shp2 with AKAP-Lbc. Both AKAP-Lbc and Shp2 are implicated in pathological cardiac hypertrophy and heart failure. We have previously demonstrated that AKAP-Lbc-anchored PKA plays a role in the induction of hypertrophy (14Carnegie G.K. Soughayer J. Smith F.D. Pedroja B.S. Zhang F. Diviani D. Bristow M.R. Kunkel M.T. Newton A.C. Langeberg L.K. Scott J.D. AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.Mol. Cell. 2008; 32: 169-179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and recent publications demonstrate that Shp2 loss of function or knock-out in mice results in hypertrophic cardiomyopathy (30Kontaridis M.I. Yang W. Bence K.K. Cullen D. Wang B. Bodyak N. Ke Q. Hinek A. Kang P.M. Liao R. Neel B.G. Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated protein kinase and RhoA signaling pathways.Circulation. 2008; 117: 1423-1435Crossref PubMed Scopus (69) Google Scholar, 31Ishida H. Kogaki S. Narita J. Ichimori H. Nawa N. Okada Y. Takahashi K. Ozono K. LEOPARD-type SHP2 mutant Gln510Glu attenuates cardiomyocyte differentiation and promotes cardiac hypertrophy via dysregulation of Akt/GSK-3β/β-catenin signaling.Am. J. Physiol. Heart Circ. Physiol. 2011; 301: H1531-H1539Crossref PubMed Scopus (29) Google Scholar, 32Marin T.M. Clemente C.F. Santos A.M. Picardi P.K. Pascoal V.D. Lopes-Cendes I. Saad M.J. Franchini K.G. Shp2 negatively regulates growth in cardiomyocytes by controlling focal adhesion kinase/Src and mTOR pathways.Circ. Res. 2008; 103: 813-824Crossref PubMed Scopus (50) Google Scholar). Therefore, we hypothesized that inhibition of Shp2 activity in the AKAP-Lbc complex by PKA may be a previously unrecognized factor in the induction of cardiac hypertrophy. To test this hypothesis, we induced cardiac hypertrophy in mice by chronic infusion of isoproterenol (25 μg/g per day for 30 days) using a minipump (33Taglieri D.M. Monasky M.M. Knezevic I. Sheehan K.A. Lei M. Wang X. Chernoff J. Wolska B.M. Ke Y. Solaro R.J. Ablation of p21-activated kinase-1 in mice promotes isoproterenol-induced cardiac hypertrophy in association with activation of ERK1/2 and inhibition of protein phosphatase 2A.J. Mol. Cell. Cardiol. 2011; 51: 988-996Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Heart morphology and function were assessed immediately by echocardiography prior to minipump implantation and after 30 days. Additionally, a small section of the left ventricle was sectioned and stained with hematoxylin. Results indicate that isoproterenol treatment significantly induced cardiac hypertrophy compared with control animals (infused with saline). Using these samples we observed that PTP activity associated with AKAP-Lbc is significantly decreased in hypertrophic heart compared with control heart (Fig. 6A and Table 1). Consistent with previous reports (14Carnegie G.K. Soughayer J. Smith F.D. Pedroja B.S. Zhang F. Diviani D. Bristow M.R. Kunkel M.T. Newton A.C. Langeberg L.K. Scott J.D. AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.Mol. Cell. 2008; 32: 169-179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 17Appert-Collin A. Cotecchia S. Nenniger-Tosato M. Pedrazzini T. Diviani D. The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates α1-adrenergic receptor-induced cardiomyocyte hypertrophy.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10140-10145Crossref PubMed Scopus (84) Google Scholar), we observed a 1.4 ± 0.1-fold increase in AKAP-Lbc expression in hypertrophic hearts; therefore, PTP activity was normalized to AKAP-Lbc expression for all IP assays. No change in Shp2 expression levels under hypertrophic conditions was observed. As in previous experiments, the levels of Shp2 co-immunoprecipitating with AKAP-Lbc were consistent in hypertrophic and control conditions, and importantly, Shp2 is phosphorylated by PKA under isoproterenol-induced hypertrophic conditions (Fig. 6B). Overall, these results suggest that chronic activation of PKA in the heart promotes the inhibition of Shp2 activity associated with AKAP-Lbc. This is a previously unrecognized mechanism that may contribute to the induction of cardiac hypertrophy (depicted in Fig. 7).TABLE 1Echocardiographic data of mice receiving saline solution or isoproterenol (ISO) for 30 daysEchocardiographic parametersBefore saline (n = 3)Control (saline) (n = 3)Before ISO (n = 3)Hypertrophic (ISO) (n = 3)HR, bpm525 ± 12512 ± 15530 ± 10595 ± 22ap < 0.05 vs. all other groups.LV FS, %31.71 ± 2.8738.24 ± 4.7735.46 ± 4.3957.42 ± 3.38ap < 0.05 vs. all other groups.EF, %59.76 ± 4.0267.82 ± 5.9363.93 ± 5.5687.56 ± 2.33ap < 0.05 vs. all other groups.LV ESV, μl61.81 ± 3.6777.86 ± 5.0855.19 ± 5.467.25 ± 1.48ap < 0.05 vs. all other groups.LV EDV, μl24.79 ± 2.8725.10 ± 2.9224.73 ± 3.3157.45 ± 5.2ap < 0.05 vs. all other groups.LV mass, mg117.50 ± 11.95125.26 ± 4.41117.50 ± 11.95177.30 ± 8.52ap < 0.05 vs. all other groups.LVAWd, mm0.87 ± 0.090.87 ± 0.060.97 ± 0.041.24 ± 0.08ap < 0.05 vs. all other groups.LVPWd, mm0.82 ± 0.050.80 ± 40.050.77 ± 0.041.11 ± 0.12ap < 0.05 vs. all other groups.a p < 0.05 vs. all other groups. Open table in a new tab FIGURE 7Model showing the role of AKAP-Lbc in the regulation of Shp2 activity by PKA. AKAP-Lbc assembles a signaling complex composed of PKA and Shp2 in cardiac myocytes. Stress conditions (e.g. chronic activation of the β-adrenergic receptor) lead to PKA activation, thereby promoting inhibition of Shp2 activity, which may contribute to the induction of cardiac hypertrophy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this report we identify the tyrosine phosphatase Shp2 as a previously unappreciated component of the AKAP-Lbc signaling complex. Our mapping experiments show that Shp2 binds predominantly to a central region of AKAP-Lbc encompassing amino acid residues 1388–1923. We show that Shp2 is a PKA substrate and that PKA phosphorylation of Shp2 inhibits its PTP activity. Furthermore, by using a mutant form of AKAP-Lbc that is unable to bind PKA, we demonstrate that AKAP-Lbc-anchored PKA modulates Shp2 phosphorylation and activity in the AKAP-Lbc complex. Because AKAP-Lbc also scaffolds and promotes the activation of PKD1, we examined whether PKD1 could phosphorylate Shp2. Our results demonstrate that although PKA and PKD1 are basophilic protein kinases with similar substrate specificity that can phosphorylate shared cardiac substrates, PKD1 does not phosphorylate Shp2. These results indicate that inhibition of Shp2 is specific to PKA signaling and does not occur downstream of PKD. We are currently mapping the site(s) of PKA phosphorylation in Shp2. By performing in vitro PKA phosphorylation reactions using fragments of Shp2, our current data indicate multiple sites of phosphorylation. Using point mutations we are investigating effects on phosphatase activity to pinpoint critical amino acid residues. Given the important cardiac roles of both AKAP-Lbc and Shp2, we performed experiments to investigate the AKAP-Lbc-Shp2 interaction in the heart. Several lines of evidence suggest that AKAP-Lbc interacts with Shp2 in the heart, which may be important for regulation of cardiac function. First, we demonstrate the co-IP of endogenous Shp2 with endogenous AKAP-Lbc from heart extract. Second, we are able to visualize this interaction using a BiFC approach, indicating a cytoplasmic Shp2-AKAP-Lbc interaction, with possible localization at the plasma membrane and association with the cytoskeleton. Third, our in vitro PTP activity assay data indicate that Shp2-PTP activity is associated with endogenous AKAP-Lbc in the heart. Mutations resulting in loss of Shp2 catalytic activity are associated with congenital heart defects and cardiac hypertrophy (23Kontaridis M.I. Swanson K.D. David F.S. Barford D. Neel B.G. PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects.J. Biol. Chem. 2006; 281: 6785-6792Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 24Stewart R.A. Sanda T. Widlund H.R. Zhu S. Swanson K.D. Hurley A.D. Bentires-Alj M. Fisher D.E. Kontaridis M.I. Look A.T. Neel B.G. Phosphatase-dependent and -independent functions of Shp2 in neural crest cells underlie LEOPARD syndrome pathogenesis.Dev. Cell. 2010; 18: 750-762Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). AKAP-Lbc is also implicated in cardiac hypertrophic signaling, and through knockdown/rescue experiments we previously showed that AKAP-Lbc-tethered PKA is important for the induction of cardiac myocyte hypertrophy (14Carnegie G.K. Soughayer J. Smith F.D. Pedroja B.S. Zhang F. Diviani D. Bristow M.R. Kunkel M.T. Newton A.C. Langeberg L.K. Scott J.D. AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.Mol. Cell. 2008; 32: 169-179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar); however, the mechanism of PKA action is unknown. To induce PKA activation and cardiac hypertrophy in vivo, mice were subjected to chronic β-adrenergic stimulation through isoproterenol infusion. Under these hypertrophic conditions, our results indicate that AKAP-Lbc-associated Shp2 activity is reduced. Thus, while induction of hypertrophy is a multifaceted process, inhibition of Shp2 activity through enhanced PKA signaling in response to chronic β-adrenergic stimulation may be a previously unrecognized mechanism promoting compensatory cardiac hypertrophy. Importantly, our Shp2 activity data using a form of AKAP-Lbc that is unable to bind PKA (AKAP-Lbc-ΔPKA) supports a model where AKAP-Lbc facilitates the phosphorylation and inhibition of Shp2 through PKA (Fig. 5). Interestingly, we and others have previously observed up-regulation of AKAP-Lbc expression under hypertrophic conditions (14Carnegie G.K. Soughayer J. Smith F.D. Pedroja B.S. Zhang F. Diviani D. Bristow M.R. Kunkel M.T. Newton A.C. Langeberg L.K. Scott J.D. AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.Mol. Cell. 2008; 32: 169-179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 17Appert-Collin A. Cotecchia S. Nenniger-Tosato M. Pedrazzini T. Diviani D. The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates α1-adrenergic receptor-induced cardiomyocyte hypertrophy.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10140-10145Crossref PubMed Scopus (84) Google Scholar). We speculate that this may facilitate the integration of PKA and Shp2 signaling in the heart. Striated muscle- and cardiac-specific Shp2-null mice show cardiac defects due to decreased ERK1/2 activation and increased Rho, Akt, and mTOR activation (30Kontaridis M.I. Yang W. Bence K.K. Cullen D. Wang B. Bodyak N. Ke Q. Hinek A. Kang P.M. Liao R. Neel B.G. Deletion of Ptpn11 (Shp2) in cardiomyocytes causes dilated cardiomyopathy via effects on the extracellular signal-regulated kinase/mitogen-activated protein kinase and RhoA signaling pathways.Circulation. 2008; 117: 1423-1435Crossref PubMed Scopus (69) Google Scholar, 34Princen F. Bard E. Sheikh F. Zhang S.S. Wang J. Zago W.M. Wu D. Trelles R.D. Bailly-Maitre B. Kahn C.R. Chen Y. Reed J.C. Tong G.G. Mercola M. Chen J. Feng G.S. Deletion of Shp2 tyrosine phosphatase in muscle leads to dilated cardiomyopathy, insulin resistance, and premature death.Mol. Cell. Biol. 2009; 29: 378-388Crossref PubMed Scopus (53) Google Scholar, 35Marin T.M. Keith K. Davies B. Conner D.A. Guha P. Kalaitzidis D. Wu X. Lauriol J. Wang B. Bauer M. Bronson R. Franchini K.G. Neel B.G. Kontaridis M.I. Rapamycin reverses hypertrophic cardiomyopathy in a mouse model of LEOPARD syndrome-associated PTPN11 mutation.J. Clin. Invest. 2011; 121: 1026-1043Crossref PubMed Scopus (191) Google Scholar); therefore, we plan to examine the effects of Shp2 inhibition by PKA on these downstream pathways. Experiments presented here were performed using a model of compensated hypertrophy, and from our echocardiography data it is clear that the hearts are not in the decompensatory phase leading to heart failure. Therefore it will be of interest to determine Shp2 activity at different stages progressing to heart failure." @default.
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• W2032020222 title "Src Homology 2 Domain-containing Phosphatase 2 (Shp2) Is a Component of the A-kinase-anchoring Protein (AKAP)-Lbc Complex and Is Inhibited by Protein Kinase A (PKA) under Pathological Hypertrophic Conditions in the Heart" @default.
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