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AKAP79 Inhibits Calcineurin through a Site Distinct from the Immunophilin-binding Region

1998; Elsevier BV; Volume: 273; Issue: 42 Linguagem: Inglês

10.1074/jbc.273.42.27412

1083-351X

Adam Kashishian, Monique Howard, Christine Loh, W. Michael Gallatin, Merl F. Hoekstra, Yvonne Y. Lai,

Peptidase Inhibition and Analysis

Targeting of protein kinases and phosphatases provides additional specificity to substrate selectivity in cellular signaling. In the case of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, AKAP79 has been shown to bind calcineurin and inhibit its activity in vitro (Coghlan, V., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Science267, 108–111). In the present study, we characterized the binding regions on calcineurin A (CnA) and AKAP79 that are important for this interaction. Residues 30–98 and 311–336 on CnA, and residues 108–280 on AKAP79 were found to be important for binding. The binding of CnA by AKAP79 does not require the calcineurin B subunit, and occurs in a region distinct from where the immunosuppressant-immunophilin complex bind. AKAP79 also bound to CnA in cells transfected with AKAP79 and CnA. To determine the function of AKAP79-calcineurin interaction in intact cells, we measured the dephosphorylation and subsequent activation of NFAT, a transcription factor that is a substrate for calcineurin. Overexpression of AKAP79 inhibited NFAT dephosphorylation, resulting in a decrease in NFAT activation. These results demonstrated that AKAP79 can bind to and inhibit calcineurin activity in vivo, suggesting a physiological role for AKAP79-calcineurin interaction in NFAT-mediated signaling. Targeting of protein kinases and phosphatases provides additional specificity to substrate selectivity in cellular signaling. In the case of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, AKAP79 has been shown to bind calcineurin and inhibit its activity in vitro (Coghlan, V., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Science267, 108–111). In the present study, we characterized the binding regions on calcineurin A (CnA) and AKAP79 that are important for this interaction. Residues 30–98 and 311–336 on CnA, and residues 108–280 on AKAP79 were found to be important for binding. The binding of CnA by AKAP79 does not require the calcineurin B subunit, and occurs in a region distinct from where the immunosuppressant-immunophilin complex bind. AKAP79 also bound to CnA in cells transfected with AKAP79 and CnA. To determine the function of AKAP79-calcineurin interaction in intact cells, we measured the dephosphorylation and subsequent activation of NFAT, a transcription factor that is a substrate for calcineurin. Overexpression of AKAP79 inhibited NFAT dephosphorylation, resulting in a decrease in NFAT activation. These results demonstrated that AKAP79 can bind to and inhibit calcineurin activity in vivo, suggesting a physiological role for AKAP79-calcineurin interaction in NFAT-mediated signaling. protein phosphatase 1 calcineurin A calcineurin B cyclosporin A A kinase anchoring proteins cAMP-dependent protein kinase A type I regulatory subunit of PKA type II regulatory subunit of PKA protein kinase C human embryonic kidney 293 cells green fluorescent protein polymerase chain reaction green lantern green fluorescent protein phorbol 12-myristate 13-acetate FK506-binding protein phosphate-buffered saline horseradish peroxidase polyacrylamide gel electrophoresis 1,4-piperazinediethanesulfonic acid. Multiple hormones signal through common second messengers to activate the same protein kinase/phosphatase cascades yet elicit distinct cellular responses (1Krebs E.G. Beavo J.A. Annu. Rev. Biochem. 1979; 48: 923-959Crossref PubMed Scopus (1407) Google Scholar). One form of regulation for these diverse effects is the intracellular compartmentalization of the kinases and phosphatases involved (2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar, 3Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (832) Google Scholar, 4Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar). For example, although ubiquitously expressed, protein phosphatase 1 (PP1)1 is localized to specific subcellular compartments, including glycogen particles in liver, neuronal dendrites, and myofibrils of skeletal muscle and smooth muscle by its association with targeting subunits (2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar,4Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar). These targeting subunits confer in vivo substrate specificity not only by localizing PP1, but also by regulating the activity of PP1 toward different substrates (2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar, 4Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar, 5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar).Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (6Klee C.B. Draeta G.P. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar), is a protein phosphatase that may be localized by targeting subunits. Calcineurin plays an essential role in many signaling pathways (6Klee C.B. Draeta G.P. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar, 7Yakel J.L. Trends Pharmacol. Sci. 1997; 18: 124-134Abstract Full Text PDF PubMed Scopus (223) Google Scholar). It consists of a catalytic A subunit (CnA), and a calcium-binding regulatory B subunit (CnB). In activated T cells, calcineurin dephosphorylates the cytoplasmic factor, NFAT, allowing it to translocate into the nucleus and activate interleukin-2 transcription (8Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar, 9Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2201) Google Scholar). Immunosuppressants, such as cyclosporin A (CsA) or FK506, when bound to their respective intracellular immunophilins, inhibit calcineurin phosphatase activity, preventing NFAT activation and subsequent cytokine gene induction (8Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar, 9Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2201) Google Scholar). Because of the diverse role calcineurin plays in cellular processes, it is likely that, similar to PP1, calcineurin targeting is an important regulatory mechanism.Insight into calcineurin targeting may also be gained from consideration of the targeting of PKA. A group of proteins, referred to as AKAPs (A-kinase-anchoringproteins), target PKA to specific microenvironments by anchoring the regulatory subunits (RI and RII) of PKA (10Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. J. Biol. Chem. 1997; 272: 8057-8064Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 11Faux M.C. Scott J.D. Cell. 1996; 85: 9-12Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 12Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Anchoring of PKA by AKAPs has been shown to be important for regulation of PKA-mediated processes (12Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). One such AKAP, AKAP79, is enriched in neurons, but is also present in T cells. AKAP79 binds membrane vesicles through its NH2 terminus (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar), and has been proposed to anchor PKA (14Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar), calcineurin (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar), and PKC (11Faux M.C. Scott J.D. Cell. 1996; 85: 9-12Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The interaction between AKAP79 and calcineurin (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar, 15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar) has been partially characterized biochemically. When calcineurin was copurified with AKAP79 from rat brain extract, calcineurin was found to be inactive in the complex. Indeed, AKAP79 inhibits purified calcineurin in vitro (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar, 15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar). However, the domains involved in the AKAP79 and calcineurin interaction are poorly defined and the functional consequence of this interaction is still unclear.In the present study, we have further characterized the regions on CnA and AKAP79 that are required for their interaction by using yeast two-hybrid analysis as well as biochemical methods. Transfection studies with wild type and mutant versions of these proteins demonstrated that AKAP79 inhibited calcineurin-dependent dephosphorylation of NFAT, suggesting that AKAP79 plays a critical role in regulating calcineurin activity in vivo.DISCUSSIONThe present study is the first to demonstrate that AKAP79 binds to CnA and inhibits calcineurin activity in vivo. The regions that are critical for binding were identified by two-hybrid analysis, by in vitro biochemical studies, and through co-immunoprecipitation experiments with transfected cells. AKAP79 was found to bind to calcineurin at sites distinct from that of the immunosuppresant-immunophilin complex, suggesting that AKAP79 and the immunosuppressants inhibit calcineurin activity by different mechanisms. The inhibition of calcineurin activity by AKAP79 was also shown in intact cells. Overexpression of AKAP79 inhibited the dephosphorylation of NFAT by endogenous calcineurin, resulting in a decrease in NFAT activation. Since NFAT activation is a key component of interleukin-2 transcriptional regulation, the inhibition of NFAT activation by AKAP79 will decrease T cell signaling.It was previously postulated, based on sequence similarity to FKBP12 and peptide inhibition studies, that residues 88–102 of AKAP79 were responsible for binding calcineurin (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar). However, we found that a peptide with the same amino acid content but with randomly scrambled sequence still inhibited calcineurin activity (data not shown). Since this peptide is highly basic, we interpreted the previous inhibition of calcineurin activity by residues 88–102 of AKAP79 (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar) to nonspecific charge interactions. Furthermore, we and others (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) have now shown that the first 108 residues of AKAP79, which contains the membrane targeting (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) and PKC-binding region (43Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (479) Google Scholar), are not required for CnA binding (Figs. 4 and 5 also Ref. 13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) or inhibition of calcineurin activity (Figs. 4 and 7 (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar)). In addition, yeast two-hybrid analysis suggests that the last 100 residues of AKAP79, containing the RII-binding region (17Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar), are also not needed for CnA binding (Fig. 4 A). Thus, PKA, PKC, and CnA bind to different regions on AKAP79 (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar, 17Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 43Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (479) Google Scholar).We have also defined the region on calcineurin required for its association with AKAP79. Two hybrid studies, as well as co-immunoprecipitation experiments showed that deletion of the first 97 or the last 152 residues of CnA abolished AKAP79 binding. Alanine scanning mutagenesis further defined the region between residues 311 and 334 as important for AKAP79 binding. These results, as well as data indicating that CnB is not required for AKAP79 binding, suggest that the AKAP79 and the immunosuppressant-immunophilin binding regions on CnA are distinct. Biochemical studies suggest that the immunosuppressant-immunophilin complex binds to the latch region of CnB (31Milan D. Griffith J. Su M. Price E.R. McKeon F. Cell. 1994; 79: 437-447Abstract Full Text PDF PubMed Scopus (98) Google Scholar), a tertiary structure that forms when CnB binds to CnA (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar, 45Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (764) Google Scholar). For AKAP79, we found that two regions are important for CnA binding, the NH2-terminal region corresponding to residues 30–96 of mouse CnA (highlighted in cyan in the three-dimensional structure of bovine CnA (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar), Fig. 9 A) and the the β13/β14 sheets formed by residues corresponding to 311–340 of mouse CnA (highlighted in purple, Fig. 9 A; sequence shown in Fig. 1 B). These two regions form a groove in the three-dimensional structure of calcineurin (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar, 45Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (764) Google Scholar) that likely contains the AKAP79-binding site (Fig. 9 A). Since PP1 serves as a model for studying the interaction between targeting subunits and protein phosphatases, we compared the three-dimensional structure of calcineurin to PP1 (Fig. 9). A peptide based on a consensus binding sequence found in many PP1 targeting subunits has been co-crystallized with PP1 (5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar). This peptide binds to a hydrophobic channel that is remote from the catalytic site. Interestingly, the COOH-terminal β13/β14 sheets in PP1 have been shown to be important contact areas for the binding of this peptide to PP1 (5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar). This hydrophobic channel is on the opposite side of the β13/β14 sheets where the corresponding AKAP79-binding region on calcineurin is. While the exact residues in the β13/β14 sheets are not conserved between CnA and PP1, the overall tertiary structure in the core region is similar (Fig. 9). Based on the deletion analysis and structural similarity, it is attractive to postulate that AKAP79 may also bind to a similar region on CnA. Co-crystallization of AKAP79 with CnA will clarify whether AKAP79 and the PP1 targeting subunits bind to similar functional domains on calcineurin and PP1, respectively.The binding of AKAP79 to calcineurin diminished calcineurin-dependent signaling in intact cells (Figs. Figure 6, Figure 7, Figure 8). Deletion of the membrane-targeting NH2-terminal 108 residues of AKAP79 did not affect its ability to inhibit calcineurin activity (Fig. 7), suggesting that AKAP79 inhibition of NFAT activation was unlikely to be due solely to AKAP79 sequestering calcineurin at the membrane, away from NFAT. This inhibitory effect of AKAP79 on calcineurin activation of NFAT also differs from that on PKA signaling to the nucleus, since anchoring of PKA by AKAP79 enhances nuclear phosphorylation of CREB (46Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The association of calcineurin with AKAP79 may represent an inactive "pool," or the association may provide a mechanism by which calcineurin-dependent signaling is regulated. Whether and how the association between AKAP79 and calcineurin is regulated remains to be determined.In addition to regulating T cell signaling, AKAP79 could also modulate calcineurin activity in other cells by targeting it to specific areas where its substrates are localized, or coordinate signaling between calcineurin and other effector molecules that interact with calcineurin (42Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 47Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1274-1277Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 48Shibasaki F. Kondo E. Akagi T. McKeon F. Nature. 1997; 386: 728-731Crossref PubMed Scopus (333) Google Scholar). In brain, AKAP79 (49Freund, T. F., Sik, A., Gulacsi, A., Katona, I., Lai, Y., Lockerbie, O., Scott, J. D., Gallatin, W. M., and Mody, I. (1997) Soc. Neurosci. Abstr. 2010Google Scholar) and calcineurin (50Sik A. Hajos N. Gulacsi A. Mody I. Freund T.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3245-3250Crossref PubMed Scopus (142) Google Scholar) are found in dendritic spines in the hippocampal pyramidal neurons where excitatory glutamate receptors are localized. Considering that calcineurin plays important roles in immune and neuronal functions, understanding the role of calcineurin targeting will provide not only a better understanding of cell-specific mechanisms of calcineurin regulation, but also a potential novel target for designing cell-specific calcineurin inhibitors. Multiple hormones signal through common second messengers to activate the same protein kinase/phosphatase cascades yet elicit distinct cellular responses (1Krebs E.G. Beavo J.A. Annu. Rev. Biochem. 1979; 48: 923-959Crossref PubMed Scopus (1407) Google Scholar). One form of regulation for these diverse effects is the intracellular compartmentalization of the kinases and phosphatases involved (2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar, 3Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (832) Google Scholar, 4Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar). For example, although ubiquitously expressed, protein phosphatase 1 (PP1)1 is localized to specific subcellular compartments, including glycogen particles in liver, neuronal dendrites, and myofibrils of skeletal muscle and smooth muscle by its association with targeting subunits (2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar,4Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar). These targeting subunits confer in vivo substrate specificity not only by localizing PP1, but also by regulating the activity of PP1 toward different substrates (2Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar, 4Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar, 5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar). Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (6Klee C.B. Draeta G.P. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar), is a protein phosphatase that may be localized by targeting subunits. Calcineurin plays an essential role in many signaling pathways (6Klee C.B. Draeta G.P. Hubbard M.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 149-200PubMed Google Scholar, 7Yakel J.L. Trends Pharmacol. Sci. 1997; 18: 124-134Abstract Full Text PDF PubMed Scopus (223) Google Scholar). It consists of a catalytic A subunit (CnA), and a calcium-binding regulatory B subunit (CnB). In activated T cells, calcineurin dephosphorylates the cytoplasmic factor, NFAT, allowing it to translocate into the nucleus and activate interleukin-2 transcription (8Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar, 9Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2201) Google Scholar). Immunosuppressants, such as cyclosporin A (CsA) or FK506, when bound to their respective intracellular immunophilins, inhibit calcineurin phosphatase activity, preventing NFAT activation and subsequent cytokine gene induction (8Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (626) Google Scholar, 9Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2201) Google Scholar). Because of the diverse role calcineurin plays in cellular processes, it is likely that, similar to PP1, calcineurin targeting is an important regulatory mechanism. Insight into calcineurin targeting may also be gained from consideration of the targeting of PKA. A group of proteins, referred to as AKAPs (A-kinase-anchoringproteins), target PKA to specific microenvironments by anchoring the regulatory subunits (RI and RII) of PKA (10Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. J. Biol. Chem. 1997; 272: 8057-8064Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 11Faux M.C. Scott J.D. Cell. 1996; 85: 9-12Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 12Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Anchoring of PKA by AKAPs has been shown to be important for regulation of PKA-mediated processes (12Dell'Acqua M.L. Scott J.D. J. Biol. Chem. 1997; 272: 12881-12884Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). One such AKAP, AKAP79, is enriched in neurons, but is also present in T cells. AKAP79 binds membrane vesicles through its NH2 terminus (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar), and has been proposed to anchor PKA (14Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar), calcineurin (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar), and PKC (11Faux M.C. Scott J.D. Cell. 1996; 85: 9-12Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The interaction between AKAP79 and calcineurin (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar, 15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar) has been partially characterized biochemically. When calcineurin was copurified with AKAP79 from rat brain extract, calcineurin was found to be inactive in the complex. Indeed, AKAP79 inhibits purified calcineurin in vitro (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar, 15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar). However, the domains involved in the AKAP79 and calcineurin interaction are poorly defined and the functional consequence of this interaction is still unclear. In the present study, we have further characterized the regions on CnA and AKAP79 that are required for their interaction by using yeast two-hybrid analysis as well as biochemical methods. Transfection studies with wild type and mutant versions of these proteins demonstrated that AKAP79 inhibited calcineurin-dependent dephosphorylation of NFAT, suggesting that AKAP79 plays a critical role in regulating calcineurin activity in vivo. DISCUSSIONThe present study is the first to demonstrate that AKAP79 binds to CnA and inhibits calcineurin activity in vivo. The regions that are critical for binding were identified by two-hybrid analysis, by in vitro biochemical studies, and through co-immunoprecipitation experiments with transfected cells. AKAP79 was found to bind to calcineurin at sites distinct from that of the immunosuppresant-immunophilin complex, suggesting that AKAP79 and the immunosuppressants inhibit calcineurin activity by different mechanisms. The inhibition of calcineurin activity by AKAP79 was also shown in intact cells. Overexpression of AKAP79 inhibited the dephosphorylation of NFAT by endogenous calcineurin, resulting in a decrease in NFAT activation. Since NFAT activation is a key component of interleukin-2 transcriptional regulation, the inhibition of NFAT activation by AKAP79 will decrease T cell signaling.It was previously postulated, based on sequence similarity to FKBP12 and peptide inhibition studies, that residues 88–102 of AKAP79 were responsible for binding calcineurin (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar). However, we found that a peptide with the same amino acid content but with randomly scrambled sequence still inhibited calcineurin activity (data not shown). Since this peptide is highly basic, we interpreted the previous inhibition of calcineurin activity by residues 88–102 of AKAP79 (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar) to nonspecific charge interactions. Furthermore, we and others (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) have now shown that the first 108 residues of AKAP79, which contains the membrane targeting (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) and PKC-binding region (43Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (479) Google Scholar), are not required for CnA binding (Figs. 4 and 5 also Ref. 13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) or inhibition of calcineurin activity (Figs. 4 and 7 (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar)). In addition, yeast two-hybrid analysis suggests that the last 100 residues of AKAP79, containing the RII-binding region (17Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar), are also not needed for CnA binding (Fig. 4 A). Thus, PKA, PKC, and CnA bind to different regions on AKAP79 (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar, 17Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 43Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (479) Google Scholar).We have also defined the region on calcineurin required for its association with AKAP79. Two hybrid studies, as well as co-immunoprecipitation experiments showed that deletion of the first 97 or the last 152 residues of CnA abolished AKAP79 binding. Alanine scanning mutagenesis further defined the region between residues 311 and 334 as important for AKAP79 binding. These results, as well as data indicating that CnB is not required for AKAP79 binding, suggest that the AKAP79 and the immunosuppressant-immunophilin binding regions on CnA are distinct. Biochemical studies suggest that the immunosuppressant-immunophilin complex binds to the latch region of CnB (31Milan D. Griffith J. Su M. Price E.R. McKeon F. Cell. 1994; 79: 437-447Abstract Full Text PDF PubMed Scopus (98) Google Scholar), a tertiary structure that forms when CnB binds to CnA (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar, 45Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (764) Google Scholar). For AKAP79, we found that two regions are important for CnA binding, the NH2-terminal region corresponding to residues 30–96 of mouse CnA (highlighted in cyan in the three-dimensional structure of bovine CnA (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar), Fig. 9 A) and the the β13/β14 sheets formed by residues corresponding to 311–340 of mouse CnA (highlighted in purple, Fig. 9 A; sequence shown in Fig. 1 B). These two regions form a groove in the three-dimensional structure of calcineurin (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar, 45Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (764) Google Scholar) that likely contains the AKAP79-binding site (Fig. 9 A). Since PP1 serves as a model for studying the interaction between targeting subunits and protein phosphatases, we compared the three-dimensional structure of calcineurin to PP1 (Fig. 9). A peptide based on a consensus binding sequence found in many PP1 targeting subunits has been co-crystallized with PP1 (5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar). This peptide binds to a hydrophobic channel that is remote from the catalytic site. Interestingly, the COOH-terminal β13/β14 sheets in PP1 have been shown to be important contact areas for the binding of this peptide to PP1 (5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar). This hydrophobic channel is on the opposite side of the β13/β14 sheets where the corresponding AKAP79-binding region on calcineurin is. While the exact residues in the β13/β14 sheets are not conserved between CnA and PP1, the overall tertiary structure in the core region is similar (Fig. 9). Based on the deletion analysis and structural similarity, it is attractive to postulate that AKAP79 may also bind to a similar region on CnA. Co-crystallization of AKAP79 with CnA will clarify whether AKAP79 and the PP1 targeting subunits bind to similar functional domains on calcineurin and PP1, respectively.The binding of AKAP79 to calcineurin diminished calcineurin-dependent signaling in intact cells (Figs. Figure 6, Figure 7, Figure 8). Deletion of the membrane-targeting NH2-terminal 108 residues of AKAP79 did not affect its ability to inhibit calcineurin activity (Fig. 7), suggesting that AKAP79 inhibition of NFAT activation was unlikely to be due solely to AKAP79 sequestering calcineurin at the membrane, away from NFAT. This inhibitory effect of AKAP79 on calcineurin activation of NFAT also differs from that on PKA signaling to the nucleus, since anchoring of PKA by AKAP79 enhances nuclear phosphorylation of CREB (46Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The association of calcineurin with AKAP79 may represent an inactive "pool," or the association may provide a mechanism by which calcineurin-dependent signaling is regulated. Whether and how the association between AKAP79 and calcineurin is regulated remains to be determined.In addition to regulating T cell signaling, AKAP79 could also modulate calcineurin activity in other cells by targeting it to specific areas where its substrates are localized, or coordinate signaling between calcineurin and other effector molecules that interact with calcineurin (42Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 47Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1274-1277Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 48Shibasaki F. Kondo E. Akagi T. McKeon F. Nature. 1997; 386: 728-731Crossref PubMed Scopus (333) Google Scholar). In brain, AKAP79 (49Freund, T. F., Sik, A., Gulacsi, A., Katona, I., Lai, Y., Lockerbie, O., Scott, J. D., Gallatin, W. M., and Mody, I. (1997) Soc. Neurosci. Abstr. 2010Google Scholar) and calcineurin (50Sik A. Hajos N. Gulacsi A. Mody I. Freund T.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3245-3250Crossref PubMed Scopus (142) Google Scholar) are found in dendritic spines in the hippocampal pyramidal neurons where excitatory glutamate receptors are localized. Considering that calcineurin plays important roles in immune and neuronal functions, understanding the role of calcineurin targeting will provide not only a better understanding of cell-specific mechanisms of calcineurin regulation, but also a potential novel target for designing cell-specific calcineurin inhibitors. The present study is the first to demonstrate that AKAP79 binds to CnA and inhibits calcineurin activity in vivo. The regions that are critical for binding were identified by two-hybrid analysis, by in vitro biochemical studies, and through co-immunoprecipitation experiments with transfected cells. AKAP79 was found to bind to calcineurin at sites distinct from that of the immunosuppresant-immunophilin complex, suggesting that AKAP79 and the immunosuppressants inhibit calcineurin activity by different mechanisms. The inhibition of calcineurin activity by AKAP79 was also shown in intact cells. Overexpression of AKAP79 inhibited the dephosphorylation of NFAT by endogenous calcineurin, resulting in a decrease in NFAT activation. Since NFAT activation is a key component of interleukin-2 transcriptional regulation, the inhibition of NFAT activation by AKAP79 will decrease T cell signaling. It was previously postulated, based on sequence similarity to FKBP12 and peptide inhibition studies, that residues 88–102 of AKAP79 were responsible for binding calcineurin (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar). However, we found that a peptide with the same amino acid content but with randomly scrambled sequence still inhibited calcineurin activity (data not shown). Since this peptide is highly basic, we interpreted the previous inhibition of calcineurin activity by residues 88–102 of AKAP79 (15Coghlan V.M. Perrino B.A. Howard M. Langeberg L.K. Hicks J.B. Gallatin W.M. Scott J.D. Science. 1995; 267: 108-111Crossref PubMed Scopus (523) Google Scholar) to nonspecific charge interactions. Furthermore, we and others (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) have now shown that the first 108 residues of AKAP79, which contains the membrane targeting (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) and PKC-binding region (43Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (479) Google Scholar), are not required for CnA binding (Figs. 4 and 5 also Ref. 13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar) or inhibition of calcineurin activity (Figs. 4 and 7 (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar)). In addition, yeast two-hybrid analysis suggests that the last 100 residues of AKAP79, containing the RII-binding region (17Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar), are also not needed for CnA binding (Fig. 4 A). Thus, PKA, PKC, and CnA bind to different regions on AKAP79 (13Dell'Acqua M.L. Faux M.C. Thorburn J. Thorburn A. Scott J.D. EMBO J. 1998; 17: 2246-2260Crossref PubMed Scopus (202) Google Scholar, 17Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 43Klauck T.M. Faux M.C. Labudda K. Langeberg L.K. Jaken S. Scott J.D. Science. 1996; 271: 1589-1592Crossref PubMed Scopus (479) Google Scholar). We have also defined the region on calcineurin required for its association with AKAP79. Two hybrid studies, as well as co-immunoprecipitation experiments showed that deletion of the first 97 or the last 152 residues of CnA abolished AKAP79 binding. Alanine scanning mutagenesis further defined the region between residues 311 and 334 as important for AKAP79 binding. These results, as well as data indicating that CnB is not required for AKAP79 binding, suggest that the AKAP79 and the immunosuppressant-immunophilin binding regions on CnA are distinct. Biochemical studies suggest that the immunosuppressant-immunophilin complex binds to the latch region of CnB (31Milan D. Griffith J. Su M. Price E.R. McKeon F. Cell. 1994; 79: 437-447Abstract Full Text PDF PubMed Scopus (98) Google Scholar), a tertiary structure that forms when CnB binds to CnA (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar, 45Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (764) Google Scholar). For AKAP79, we found that two regions are important for CnA binding, the NH2-terminal region corresponding to residues 30–96 of mouse CnA (highlighted in cyan in the three-dimensional structure of bovine CnA (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar), Fig. 9 A) and the the β13/β14 sheets formed by residues corresponding to 311–340 of mouse CnA (highlighted in purple, Fig. 9 A; sequence shown in Fig. 1 B). These two regions form a groove in the three-dimensional structure of calcineurin (44Kissinger C.R. Parge H.E. Knighton D.R. Lewis C.T. Pelletier L.A. Tempczyk A. Kalish V.J. Tucker K.D. Showalter R.E. Moomaw E.W. Nature. 1995; 378: 641-644Crossref PubMed Scopus (690) Google Scholar, 45Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (764) Google Scholar) that likely contains the AKAP79-binding site (Fig. 9 A). Since PP1 serves as a model for studying the interaction between targeting subunits and protein phosphatases, we compared the three-dimensional structure of calcineurin to PP1 (Fig. 9). A peptide based on a consensus binding sequence found in many PP1 targeting subunits has been co-crystallized with PP1 (5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar). This peptide binds to a hydrophobic channel that is remote from the catalytic site. Interestingly, the COOH-terminal β13/β14 sheets in PP1 have been shown to be important contact areas for the binding of this peptide to PP1 (5Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (530) Google Scholar). This hydrophobic channel is on the opposite side of the β13/β14 sheets where the corresponding AKAP79-binding region on calcineurin is. While the exact residues in the β13/β14 sheets are not conserved between CnA and PP1, the overall tertiary structure in the core region is similar (Fig. 9). Based on the deletion analysis and structural similarity, it is attractive to postulate that AKAP79 may also bind to a similar region on CnA. Co-crystallization of AKAP79 with CnA will clarify whether AKAP79 and the PP1 targeting subunits bind to similar functional domains on calcineurin and PP1, respectively. The binding of AKAP79 to calcineurin diminished calcineurin-dependent signaling in intact cells (Figs. Figure 6, Figure 7, Figure 8). Deletion of the membrane-targeting NH2-terminal 108 residues of AKAP79 did not affect its ability to inhibit calcineurin activity (Fig. 7), suggesting that AKAP79 inhibition of NFAT activation was unlikely to be due solely to AKAP79 sequestering calcineurin at the membrane, away from NFAT. This inhibitory effect of AKAP79 on calcineurin activation of NFAT also differs from that on PKA signaling to the nucleus, since anchoring of PKA by AKAP79 enhances nuclear phosphorylation of CREB (46Feliciello A. Li Y. Avvedimento E.V. Gottesman M.E. Rubin C.S. Curr. Biol. 1997; 7: 1011-1014Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The association of calcineurin with AKAP79 may represent an inactive "pool," or the association may provide a mechanism by which calcineurin-dependent signaling is regulated. Whether and how the association between AKAP79 and calcineurin is regulated remains to be determined. In addition to regulating T cell signaling, AKAP79 could also modulate calcineurin activity in other cells by targeting it to specific areas where its substrates are localized, or coordinate signaling between calcineurin and other effector molecules that interact with calcineurin (42Loh C. Shaw K.T. Carew J. Viola J.P. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 47Wesselborg S. Fruman D.A. Sagoo J.K. Bierer B.E. Burakoff S.J. J. Biol. Chem. 1996; 271: 1274-1277Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 48Shibasaki F. Kondo E. Akagi T. McKeon F. Nature. 1997; 386: 728-731Crossref PubMed Scopus (333) Google Scholar). In brain, AKAP79 (49Freund, T. F., Sik, A., Gulacsi, A., Katona, I., Lai, Y., Lockerbie, O., Scott, J. D., Gallatin, W. M., and Mody, I. (1997) Soc. Neurosci. Abstr. 2010Google Scholar) and calcineurin (50Sik A. Hajos N. Gulacsi A. Mody I. Freund T.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3245-3250Crossref PubMed Scopus (142) Google Scholar) are found in dendritic spines in the hippocampal pyramidal neurons where excitatory glutamate receptors are localized. Considering that calcineurin plays important roles in immune and neuronal functions, understanding the role of calcineurin targeting will provide not only a better understanding of cell-specific mechanisms of calcineurin regulation, but also a potential novel target for designing cell-specific calcineurin inhibitors. We thank Tony DeMaggio at ICOS for analyzing the three-dimensional structure of calcineurin and PP1 and for preparing Fig. 9; Bart Steiner for preparing many of the constructs for protein expression; Drs. John D Scott, Mark L. Dell'Acqua, and Marie C. Faux at Vollum Institute, Angus C. Nairn at Rockefeller University, and Kenneth Mackie at University of Washington for critically reading the manuscript; and Alice Dersham for preparing the manuscript.