Identification of the MAP2- and P75-binding domain in the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase: Cloning and expression of the cDNA for bovine brain RIIβ

Zhijun Lu, Bridget Shafit-Zagardo, Jack Erlichman

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Abstract

cDNA clones coding for the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in λgt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RIIβ proteins. A series of expression vectors coding for truncated RIIβ proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RIIβ-fusion proteins were used in overlay assays to identify the region in RIIβ which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C. S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RIIβ NH2 terminus (RIIβ(1-50)) bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H. M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RIIβ-fusion proteins containing RIIβ amino acids 1-50. Three fusion proteins, RIIβ(1-25), RIIβ(25-96), and RIIβ(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RIIβ. Preincubation of bovine heart protein kinase IIα and RIIβ(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.

Original languageEnglish (US)
Pages (from-to)21804-21810
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number35
StatePublished - Dec 15 1990

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Microtubule-Associated Proteins
Cloning
Cyclic AMP-Dependent Protein Kinases
Organism Cloning
Brain
Complementary DNA
Fusion reactions
Proteins
Amino Acids
Collodion
Binding Sites
Calmodulin-Binding Proteins
Protein Kinases
Escherichia coli
Gene Library
Neurons
Rats
Assays
Plasmids
Monoclonal Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{fb786c4e1d6348fe995652d03e15f6a9,
title = "Identification of the MAP2- and P75-binding domain in the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase: Cloning and expression of the cDNA for bovine brain RIIβ",
abstract = "cDNA clones coding for the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in λgt11. The cDNA codes for a protein of 418 amino acids which is 98{\%} homologous to the rat and human RIIβ proteins. A series of expression vectors coding for truncated RIIβ proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RIIβ-fusion proteins were used in overlay assays to identify the region in RIIβ which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C. S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RIIβ NH2 terminus (RIIβ(1-50)) bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H. M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RIIβ-fusion proteins containing RIIβ amino acids 1-50. Three fusion proteins, RIIβ(1-25), RIIβ(25-96), and RIIβ(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RIIβ. Preincubation of bovine heart protein kinase IIα and RIIβ(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.",
author = "Zhijun Lu and Bridget Shafit-Zagardo and Jack Erlichman",
year = "1990",
month = "12",
day = "15",
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volume = "265",
pages = "21804--21810",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
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TY - JOUR

T1 - Identification of the MAP2- and P75-binding domain in the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase

T2 - Cloning and expression of the cDNA for bovine brain RIIβ

AU - Lu, Zhijun

AU - Shafit-Zagardo, Bridget

AU - Erlichman, Jack

PY - 1990/12/15

Y1 - 1990/12/15

N2 - cDNA clones coding for the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in λgt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RIIβ proteins. A series of expression vectors coding for truncated RIIβ proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RIIβ-fusion proteins were used in overlay assays to identify the region in RIIβ which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C. S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RIIβ NH2 terminus (RIIβ(1-50)) bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H. M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RIIβ-fusion proteins containing RIIβ amino acids 1-50. Three fusion proteins, RIIβ(1-25), RIIβ(25-96), and RIIβ(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RIIβ. Preincubation of bovine heart protein kinase IIα and RIIβ(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.

AB - cDNA clones coding for the regulatory subunit (RIIβ) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in λgt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RIIβ proteins. A series of expression vectors coding for truncated RIIβ proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RIIβ-fusion proteins were used in overlay assays to identify the region in RIIβ which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C. S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RIIβ NH2 terminus (RIIβ(1-50)) bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H. M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RIIβ-fusion proteins containing RIIβ amino acids 1-50. Three fusion proteins, RIIβ(1-25), RIIβ(25-96), and RIIβ(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RIIβ. Preincubation of bovine heart protein kinase IIα and RIIβ(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.

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