Interaction of Phosphorylated Tryptophan Hydroxylase with 14-3-3 Proteins

Abstract

Rabbit brain tryptophan hydroxylase (TPH) has been expressed in insect cells (Spodoptera frugiperda) as a histidine-tagged enzyme. The specific activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryptophan/min/mg. Multifunctional regulatory 14-3-3 proteins were purified from fresh bovine brain. Phosphorylation and 14-3-3 proteins play important roles in the regulation of TPH activity. We have found that phosphorylation of TPH by cAMP-dependent protein kinase increased the activity of the hydroxylase by 25–30% and that 14-3-3 proteins increased the hydroxylase activity of phosphorylated TPH by ∼45%. Under these conditions, the 14-3-3 proteins were not phosphorylated, and unphosphorylated TPH was not activated by 14-3-3 proteins. Surface plasmon resonance analysis demonstrated that 14-3-3 proteins bind to phosphorylated TPH with an affinity constant (K a ) of 4.5 × 107m−1. Binding studies using affinity chromatography also showed that 14-3-3 proteins interact with phosphorylated TPH. The dephosphorylation of TPH by protein phosphatase-1 was inhibited by 14-3-3 proteins. Our results demonstrate that 14-3-3 proteins form a complex with phosphorylated brain TPH, thereby increasing its enzymatic activity and inhibiting its dephosphorylation. Rabbit brain tryptophan hydroxylase (TPH) has been expressed in insect cells (Spodoptera frugiperda) as a histidine-tagged enzyme. The specific activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryptophan/min/mg. Multifunctional regulatory 14-3-3 proteins were purified from fresh bovine brain. Phosphorylation and 14-3-3 proteins play important roles in the regulation of TPH activity. We have found that phosphorylation of TPH by cAMP-dependent protein kinase increased the activity of the hydroxylase by 25–30% and that 14-3-3 proteins increased the hydroxylase activity of phosphorylated TPH by ∼45%. Under these conditions, the 14-3-3 proteins were not phosphorylated, and unphosphorylated TPH was not activated by 14-3-3 proteins. Surface plasmon resonance analysis demonstrated that 14-3-3 proteins bind to phosphorylated TPH with an affinity constant (K a ) of 4.5 × 107m−1. Binding studies using affinity chromatography also showed that 14-3-3 proteins interact with phosphorylated TPH. The dephosphorylation of TPH by protein phosphatase-1 was inhibited by 14-3-3 proteins. Our results demonstrate that 14-3-3 proteins form a complex with phosphorylated brain TPH, thereby increasing its enzymatic activity and inhibiting its dephosphorylation. Tryptophan hydroxylase (TPH 1The abbreviations used are: TPH, tryptophan hydroxylase; TEV, tobacco etch virus; NTA, nitrilotriacetic acid; PKA, cAMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; 5-HTP, 5-hydroxytryptophan; SPR, surface plasmon resonance; RU, resonance unit(s). 1The abbreviations used are: TPH, tryptophan hydroxylase; TEV, tobacco etch virus; NTA, nitrilotriacetic acid; PKA, cAMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; 5-HTP, 5-hydroxytryptophan; SPR, surface plasmon resonance; RU, resonance unit(s).; EC 1.14.16.4), a member of the family of pterin-dependent aromatic amino-acid hydroxylases, catalyzes the conversion ofl-tryptophan to 5-hydroxy-l-tryptophan (1Lovenberg W. Jequier E. Sjoerdsma A. Science. 1967; 155: 217-219Crossref PubMed Scopus (371) Google Scholar, 2Kaufman S. Fisher D.B. Hayaishi O. Molecular Mechanisms of Oxygen Activation. 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Chem. 1980; 255: 4137-4143Abstract Full Text PDF PubMed Google Scholar, 8Nakata H. Fujisawa H. Eur. J. Biochem. 1982; 122: 41-47Crossref Scopus (70) Google Scholar). TPH isolated from different sources also exhibits distinct properties (8Nakata H. Fujisawa H. Eur. J. Biochem. 1982; 122: 41-47Crossref Scopus (70) Google Scholar, 9Yang X.-J. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6659-6663Crossref PubMed Scopus (48) Google Scholar). Rabbit brain TPH expressed in Escherichia coli is largely insoluble (10Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar) and extremely unstable (11Vrana K.E. Rucker P.J. Kumer S.C. Life Sci. 1994; 55: 1045-1052Crossref PubMed Scopus (30) Google Scholar). Moreover, little is known about its regulatory properties in vitro, except that it is sensitive to substrate inhibition (12Friedman P.A. Kappelman A.H. Kaufman S. J. Biol. 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Harrington M.G. N. Engl. J. Med. 1996; 335: 924-930Crossref PubMed Scopus (628) Google Scholar). Although the exact function of 14-3-3 is not known, numerous biological activities have been attributed to it. Given the large and diverse numbers of proteins that can apparently bind 14-3-3, it is important to determine the nature of these interactions. Several observations suggest that 14-3-3 interactions involve binding to phosphoserine. Activation of tyrosine hydroxylase by 14-3-3 requires prior phosphorylation of the hydroxylase with the serine/threonine kinase calmodulin kinase II (24Yamauchi T. Nakata H. Fujisawa H. J. Biol. Chem. 1981; 256: 5404-5409Abstract Full Text PDF PubMed Google Scholar). Phosphatase treatment of Raf-1 and Bcr inhibits their association with 14-3-3 in vitro (25Michaud N.R. Fabian J.R. Mathes K.D. Morrison D.K. Mol. Cell. Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (189) Google Scholar), and 14-3-3 binding to Raf-1 blocks the ability of phosphatases to inhibit Raf-1 activity (26Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (172) Google Scholar). Furthermore, 14-3-3 may bind to phosphoserine in a sequence-specific manner (57Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar). Several reports have described the bacterial expression of TPHs from different species (9Yang X.-J. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6659-6663Crossref PubMed Scopus (48) Google Scholar, 10Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar, 27Kim K.S. Wessel T.C. Stone D.M. Carver C.H. Joh T.H. Park D.H. Mol. Brain Res. 1991; 9: 277-283Crossref PubMed Scopus (67) Google Scholar). However, the bacterially expressed enzymes have proven to be mostly insoluble. By use of a fusion expression system, soluble TPH species have been expressed in E. coli (8Nakata H. Fujisawa H. Eur. J. Biochem. 1982; 122: 41-47Crossref Scopus (70) Google Scholar, 28Stoll J. Kozak C.A. Goldman D. Genomics. 1990; 7: 88-96Crossref PubMed Scopus (63) Google Scholar). It has been reported for rat phenylalanine and tyrosine hydroxylases that the baculovirus expression system yields recombinant enzymes with properties that are more similar to those of the native enzymes than these hydroxylases expressed in E. coli (29Fitzpatrick P.F. Chlumsky L.J. Daubner S.C. O'Malley K.L. J. Biol. Chem. 1990; 265: 2042-2047Abstract Full Text PDF PubMed Google Scholar, 30Wang Y.H. Citron B. Ribeiro P. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8779-8783Crossref PubMed Scopus (33) Google Scholar). Based on these observations, we decided that the baculovirus-insect cell expression system might provide an approach to obtaining soluble active TPH. This the expression of brain histidine-tagged TPH in insect unphosphorylated and phosphorylated we have the and binding of TPH to 14-3-3 proteins as as the of this on and dephosphorylation of TPH. The baculovirus expression and recombinant were from Life The bovine brain of was from and the was from The was from were of The baculovirus expression system was used for expression of brain TPH. The TPH was from (10Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar) by reaction using the and and was the by the TPH was with a with and and the and of the of the recombinant in E. coli recombinant were by of the The recombinant was isolated and used for of insect cells using for the was to a of × of brain TPH was by cells a of of for in the of were by with and were in a The cell were in of of of of and were by for The was a × with of a of The was with of and and with of and were on TPH and the of the active of TPH was by and The was using recombinant to the 14-3-3 proteins were purified to a described Biochem. J. 1992; PubMed Scopus Google Scholar) with bovine brain was in of DTT, and The was × for The was to by a × 80 with and and a of 14-3-3 proteins were a × with E. with of a of in was a of 14-3-3 proteins were to a × with and a of 14-3-3 proteins from the were on a The used was in a of and were showed that the 14-3-3 proteins were on the of 14-3-3 on the activity of TPH were with histidine-tagged enzyme. TPH activity was by the of the as described Kaufman S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) with a The DTT, and in a of The reaction was by the of TPH, for with of and on for and for the of the was on an using as the of TPH activity is as the of TPH that catalyzes the of nmol of Phosphorylation of TPH was in a reaction DTT, and of TPH. The reaction was by the of of and for The phosphorylation reaction was by the of a of the TPH was phosphorylated with and in the and of 14-3-3 proteins for in The phosphorylation reaction was with a of the The dephosphorylation reaction was by the of of protein phosphatase-1 The reaction was different by the of using bovine as a protein for the was in and the of the protein was TPH was in and and phosphorylated TPHs were on and of 14-3-3 proteins were the complex was with and proteins were by The of phosphorylated TPH with 14-3-3 was by surface plasmon resonance using Inc., The and have been B. S. G. Biochem. 1991; PubMed Scopus Google Scholar, U. B. B. R. K. S. B. H. 1991; Scholar). binding were with recombinant TPH The used was DTT, and The 14-3-3 proteins were to the on a using an the The were using the The of the complex can be as the resonance binding with P. M. C. S.J. 1994; Scopus Google Scholar, W. Goldman E. 1996; PubMed Scopus Google Scholar), the of can be to the it is The of on the surface of the is from the of the complex and the binding of the surface as a we the a a the association constant (K a ) was from the different by for The constant (K ) was also from the of of association by analysis mediated by was by Kaufman S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were by the were using expression and to affinity of active TPH, a fusion protein expression and system was used by of of an expression a baculovirus in E. coli Lee S.C. J. 1993; PubMed Google Scholar) as described The fusion enzyme a affinity a and the for of the from the The of insect cells with recombinant baculovirus was using different of and also by of the of a constant of that a of of in a a expression of soluble active TPH. The specific activity of TPH in the was of Because of the of TPH H. A. PubMed Scopus Google Scholar), was in the to the enzyme. the expressed TPH and the purified TPH the of the fusion enzyme was and were recombinant The activities of the purified fusion enzyme and the TPH were the nmol of of This is to that found for TPH with protein (9Yang X.-J. Kaufman S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6659-6663Crossref PubMed Scopus (48) Google Scholar). The was and the was The 14-3-3 proteins were from bovine brain as described similar to those described and of Scholar, P.F. P. R.J. J. 1982; PubMed Scopus Google Scholar). The 14-3-3 proteins isolated from are more species P.F. P. R.J. J. 1982; PubMed Scopus Google Scholar). with this 14-3-3 was by with of and in a of TPH was phosphorylated by using of of of TPH was a phosphorylation The and of TPH phosphorylation by were not by 14-3-3 proteins. The of of of TPH is the by (24Yamauchi T. Nakata H. Fujisawa H. J. Biol. Chem. 1981; 256: 5404-5409Abstract Full Text PDF PubMed Google Scholar). This be to a different of TPH used in the In an initial the ability of different of 14-3-3 to phosphorylated TPH was shown in the 14-3-3 activated phosphorylated TPH with 14-3-3 proteins was of 14-3-3 on the activity of unphosphorylated TPH, as can be in the in was by bovine The of recombinant TPH by 14-3-3 proteins is than the with purified TPH from rat Y. S. Fujisawa H. FEBS Lett. 1990; 268: PubMed Scopus Google Scholar). The of by TPH different is shown in TPH was phosphorylated as described TPH in the of bovine was in the as phosphorylated TPH, the first of 14-3-3 the activity of phosphorylated TPH by Under the of this in the of 14-3-3, of TPH phosphorylation a in hydroxylase activity The of phosphorylation and 14-3-3 proteins in a of TPH activity The results also that the in the was in TPH activity to phosphorylation 14-3-3 proteins. In the shown in 14-3-3 proteins were the phosphorylation was 14-3-3 proteins were the phosphorylation determine 14-3-3 proteins the of phosphorylated TPH, we first phosphorylated the enzyme and the reaction as described This phosphorylated enzyme was for of in the and of 14-3-3 proteins. a in the of 14-3-3 was a of in the of 14-3-3 was a of activity. results are the first that of the of TPH to 14-3-3 may be to a of This of 14-3-3 is similar to the of 14-3-3 proteins on tyrosine hydroxylase phosphorylated by protein kinase II C. J. B. J. J. Eur. J. Biochem. 1993; PubMed Scopus Google Scholar). The results that 14-3-3 proteins might phosphorylated TPH from by its dephosphorylation. this we the of 14-3-3 on the dephosphorylation TPH by protein TPH was with protein phosphatase-1 in the of 14-3-3 proteins shown in 14-3-3 proteins the phosphorylated enzyme dephosphorylation. In dephosphorylation in the of This inhibition of TPH dephosphorylation by 14-3-3 not from 14-3-3 proteins inhibiting protein phosphatase-1 as the dephosphorylation of proteins by protein phosphatase-1 is not inhibited by 14-3-3 proteins (26Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (172) Google Scholar, D. J. C. Collier R.J. Fu H. R. 1995; PubMed Scopus Google Scholar). We 14-3-3 proteins can interact with TPH in a manner TPH was on its and with 14-3-3 proteins. In a the TPH was with 14-3-3 proteins. The enzyme and were with to 14-3-3 and with in to TPH and 14-3-3 proteins 14-3-3 proteins to the and 14-3-3 to bind to unphosphorylated TPH and phosphorylated TPH more 14-3-3 to phosphorylated TPH than to unphosphorylated TPH. the of the we that of phosphorylated TPH of 14-3-3 and that of unphosphorylated TPH of 14-3-3 The binding of phosphorylated TPH to 14-3-3 was also using The B. S. G. Biochem. 1991; PubMed Scopus Google Scholar) an to in the a The binding of protein to the the the and it is this that is and in resonance This binding in and the of association and to be The 14-3-3 proteins were to the surface and for their ability to bind phosphorylated TPH. The in the of phosphorylated TPH with 14-3-3 proteins. TPH a in The association was by the in TPH and the was from the in of the TPH the surface was with the on was to be × was to be × The constant (K ) from the on was in was the was and and were not by a in the of for phosphorylated TPH was also from the of resonance TPH for binding was to be The shown in were using 14-3-3 proteins than those shown in A. The is shown in The a from the of the was to be 4.5 × 107m−1. The characterization of TPH and its with 14-3-3 proteins have been hampered by low yields of TPH from and the of TPH expressed in (10Tipper J.P. Citron B.A. Ribeiro P. Kaufman S. Arch. Biochem. Biophys. 1994; 315: 445-453Crossref PubMed Scopus (24) Google Scholar, K.E. Rucker P.J. Kumer S.C. Life Sci. 1994; 55: 1045-1052Crossref PubMed Scopus (30) Google Scholar, P.A. Kappelman A.H. Kaufman S. J. Biol. Chem. 1972; 247: 4165-4173Abstract Full Text PDF PubMed Google Scholar). In this we these by using a baculovirus expression Rabbit brain fusion TPH was expressed in this system, and the enzyme was purified by affinity chromatography on The purified soluble and active TPH showed a protein on with a of native brain TPH (12Friedman P.A. Kappelman A.H. Kaufman S. J. Biol. Chem. 1972; 247: 4165-4173Abstract Full Text PDF PubMed Google Scholar, S. Scopus Google Scholar), brain recombinant histidine-tagged TPH exhibits with inhibition by in the of not It has been that in the of phenylalanine hydroxylase D.B. Kaufman S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and tyrosine hydroxylase R. Kaufman S. 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The of enzyme used in that was and a was used to the We a low of 14-3-3 proteins binding to unphosphorylated TPH, of 14-3-3 of unphosphorylated TPH This be to the TPH expressed in baculovirus phosphorylated to a low affinity unphosphorylated TPH and 14-3-3 proteins. The binding of phosphorylated TPH to 14-3-3 and its were using We a of by is in with the affinity constant (K a 4.5 × from the The for with is in with the of 14-3-3 proteins to phosphorylated TPH We the of binding unphosphorylated TPH and 14-3-3 proteins as by the in The for unphosphorylated TPH not a binding from have the of with a in protein properties by can be by this have the that of the proteins be This the binding the we histidine-tagged TPH the we little 14-3-3 However, of proteins has been for different M. M. M. 1996; PubMed Scopus Google Scholar, W. PubMed Scopus Google Scholar). The by this for the binding of 14-3-3 proteins to TPH are similar to those for the binding of to Raf-1 (57Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar). it is in TPH is the of 14-3-3 in TPH are in and analysis by would to be a to determine in binding The binding for 14-3-3 binding is (57Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar). Our that are in TPH that can be phosphorylated, and and that is more by than has also been as the of phosphorylation D.M. R. J.C. J. PubMed Scopus Google Scholar). results suggest that in the in 14-3-3 proteins the dephosphorylation of phosphorylated TPH, the binding may involve than However, the not to the We are The 14-3-3 family of proteins has important in as as soluble protein have been P.F. P. R.J. J. 1982; PubMed Scopus Google Scholar). However, physiological function been attributed to 14-3-3 it was shown to be to an protein of tyrosine hydroxylase and TPH T. Isobe T. Okuyama T. Yamauchi T. 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Biol. 1995; 15: 3390-3397Crossref PubMed Scopus (189) Google Scholar) and Raf-1 (26Dent P. Jelinek T. Morrison D.K. Weber M.J. Sturgill T.W. Science. 1995; 268: 1902-1906Crossref PubMed Scopus (172) Google Scholar) inhibits their binding to 14-3-3 proteins. The dephosphorylation of F. FEBS Lett. 1994; PubMed Scopus Google Scholar), Raf-1 (57Muslin A.J. Tanner J.W. Allen P.M. Shaw A.S. Cell. 1996; 84: 889-897Abstract Full Text Full Text PDF PubMed Scopus (1185) Google Scholar), and the phosphorylated from G. P. N. M. A. C. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) is inhibited by 14-3-3 proteins. Although the 14-3-3 proteins have been shown to be an important for the protein kinase of tyrosine hydroxylase and TPH, we are a for the first for the of 14-3-3 in the and binding of phosphorylated TPH. The binding of 14-3-3 to phosphorylated TPH to have it to the phosphorylated form of TPH and its hydroxylase activity. it TPH dephosphorylation by its by of the to the phosphorylated Because unphosphorylated TPH is than phosphorylated TPH, 14-3-3 proteins binding to phosphorylated TPH in the cell would be to TPH by its dephosphorylation by The regulation of TPH by phosphorylation and binding to 14-3-3 proteins may be a The dephosphorylation may in an in activity and of the enzyme to is to the in TPH that on phosphorylation interact with 14-3-3 proteins and hydroxylase activity. It is also to the and that the of TPH. We for on this We also for and for insect