398
PROTEIN PHOSPHATASES
[34]
identity) than PP1 ( - 4 0 % identity). 6,46 It is clearly essential to study the enzymatic properties of these novel enzymes, either by isolating them or expressing the cDNA clones, in order to determine whether they can be classified as type 1 or type 2A, or whether their properties are so distinctive that additions to the current nomenclature are required.
[34] C l o n i n g o f P r o t e i n - S e r i n e / T h r e o n i n e
Phosphatases
By PATRICIA T. W. COHEN Four major types of protein-serine/threonine phosphatase (PP) catalytic subunit (PP1, PP2A, PP2B, and PP2C) have been identified in mammalian cells on the basis of their enzymatic properties.~ Over the past few years cDNAs encoding each of these enzymes have been cloned by constructing suitable oligonucleotides to peptide sequences derived from the purified phosphatases, and employing them in hybridization studies to isolate appropriate clones from bacteriophage cDNA libraries. Since these approaches have been used to clone many other proteins, they will be described fairly briefly in this chapter with emphasis on aspects of the methodology that the author has found particularly useful or are not documented clearly elsewhere. Subsequent library screening with the cDNAs encoding PP1 and PP2A led to the isolation of isoforms, homologs from different species, and cDNAs encoding putative protein phosphatases that had not been detected previously by the techniques of protein chemistry and enzymology. Procedures for detecting such novel enzymes are also discussed in this chapter, because additional phosphatases related to PP1 and PP2A are likely to be present in eukaryotic cells. Procedures for Cloning of PP1 and PP2A
Isolation of Peptides for Sequence Analysis PP1 and PP2A are low-abundance proteins composing 3000 Ci/ mmol) in a standard reaction mixture, 9 proceeds virtually to completion, and removal of unincorporated ATP is unnecessary.
Screening of Bacteriophage Libraries Libraries in bacteriophage are utilized in order that large numbers of cDNA clones ( 1 0 5 - 1 0 6 pfu) can be rapidly screened. Since peptide sequences of PP1 and PP2A had been obtained from the rabbit skeletal muscle enzymes, cDNA libraries are constructed in phage hgtl0 ~° from rabbit skeletal muscle and liver RNA using cDNA synthesized by the method of Gubler and Hoffman,ll or purchased from Clontech (Palo Alto, CA). Libraries are then screened by hybridization after in situ amplification of bacteriophage plaques. ~2All materials and procedures not described
6 y . Takahashi, K. Kato, Y. Hayashizaki, T. Wakabayashi, E. Ohtsuka, S. Matsuki, M. Ikehara, and K. Matsubara, Proc. Natl. Acad. Sci. U.S.A. 82, 1931 (1985). 7 F. H. Martin and M. M. Castro, Nucleic Acids Res. 13, 8927 (1985). 8 D. Guerini and C. Klee, Proc. Natl. Acad. Sci. U.S.A. 86, 9183 (1989). 9 R. B. Wallace and C. G. Miyada, this series, Vol. 152, p. 432. l0 T. V. Huynh, R. A. Young, and R. W. Davis, in " D N A Cloning: A Practical Approach" (D. M. Glover, ed.), Vol. 1, p. 49. IRL Press, Oxford, 1985. n U. Gubler and B. J. Hoffman, Gene 25, 263 (1983). ~2 S. L. C. Woo, this series, Vol. 68, p. 389.
[34]
CLONING OF PROTEIN-SERINE/THREONINE
PHOSPHATASES
401
in detail below can be found in Maniatis et al. 13The following modifications proved to be effective. 1. To encourage preservation of poorly growing clones, recombinant hgtl0 phage and host Escherichia coli C600 Hfl cells (Clontech) are plated on LB plates containing 10 mM MgSO4, since Mg2÷ helps stabilize phage heads. In addition, -85% identity), as are the isoforms of PP2A (>95% identity), virtually all the differences being confined to the N and C termini. In Drosophila, three isoforms of PP1 have been identified from their cDNAs. 3° Two of the Drosophila PP1 isoforms are very similar to mammalian P P l a , while the other would appear to be the homolog of mammalian PPlfl. An a-like PP1 (dis2), as well as a further isoform (sds21), are also present in Schizosaccharomyces p o m b e . 39 c D N A cloning of PP2B from mammalian brain has revealed two isoforms (PP2Ba and PP2B/3) which are the products of separate genes and show 82% overall identity. El Two further forms of PP2Ba 4° and three of PP2Bfl 8,41 can be generated by alternative splicing at the 3' end, and, in the case of one PP2B/3 form, additional splicing near the 5' end. 8 The structure of one isoform of PP2C (termed PP2Ca) has been determined by c D N A cloning, z2 PP2Ca and PP2C/3 have been purified from both rabbit skeletal muscle and rabbit liver and showed 80% amino acid sequence identity over the 62 residues where they could be compared directly.4Z Two Distinct Protein-Serine/Threonine Phosphatase Gene Families
The amino acid sequences deduced from the cDNAs of mammalian PP I and PP2A show 41% overall identity, indicating that they are members of the same gene family. The most divergent regions are the N and C termini, and the identity is 49% in the catalytic domain (Table I). The novel protein phosphatases PPX, 25 ppy,26 ppz25 and PPV, 25 and a yeast phosphatase, termed SIT4, 43 have similar domain structures to PP1 and PP2A and their identities to PP1 and PP2A are summarized in Table I. PPY and PPZ are more similar to PP1, while PPX, PPV, and SIT4 resemble PP2A more closely. PP2B is a member of the same gene family, but a more 39H. Ohkura, N. Kinoshita, S. Migatani, T. Toda, and M. Yanagida, Cell (Cambridge, Mass. ) 57, 997 (1989). 40R. L. Kincaid, R. Rathna Giri, S. Higuchi, J. Tamura, S. C. Dixon, C. A. Marietta, D. A. Amorese, and B. M. Martin, J. Biol. Chem. 265, 11312(1990). 41A. E. McPartlin, H. M. Barker, and P. T. W. Cohen, Biochim. Biophys. Acta, 1088, 308 (1991). 42C. H. McGowan, Ph.D. Thesis, University of Dundee (1988). 43K. T. Arndt, C. A. Styles, and G. R. Fink, Cell (Cambridge, Mass.) 56, 527 (1989).
406
PROTEIN PHOSPHATASES
.¢
2
¢q
o
e~
[34]
.'~.
oo
eq Z g]
"
go oq
< Z ¢) . .
0
"
go
o
Z go
.-t2. .1 e~
.
1 l>l :>1 ~1 I>l I>1 I~ I~ I¢I E-I E~ E-I e'l E'I l~
E ~ = o'l::
~m(
'W gle¢
~'r"
~
~
0
..
o
408
PROTEIN PHOSPHATASES
[35]
distantly related one. Its catalytic domain shows less similarity to PP1 and PP2A, and in addition it possesses two insertions in the catalytic domain as well as a long C-terminal extension that is involved in interaction with calmodulin. Bacteriophage h ORF221 is the shortest protein phosphatase known (221 amino acids) and the most divergent member of this family (Table 1). 36'37 Although the overall sequence identity is low (Table I), the Nterminal half of ORF221 shows 35% identity to PP1 or PP2A and includes the three highly conserved sections, corresponding to residues 63-67, 90-97, and 121-126 of P P l a (Fig. I). It is presumed that these regions are part of the active site. Oligonucleotides synthesized to these most highly conserved regions may prove useful for cloning protein phosphatases in this family that are highly divergent from PP1 and PP2A. This strategy has been successful for protein kinases. 44:5 PP2C, investigated both by peptide sequencing42 and cDNA cloning; 22 has no discernable sequence similarities to the PP1/PP2A/PP2B/hORF221 family. Thus it is a member of a quite distinct protein-serine/threonine phosphatase gene family. 44 S. K. Hanks and R. A. Lindberg, this series, Vol. 200, p. 525. 45 A. E. Wilks, this series, Vol. 200, p. 533.
[35] R e a c t i v a t i o n o f P r o t e i n P h o s p h a t a s e 1 E x p r e s s e d at High Levels from Recombinant Baculovirus
By PATRICIA T. W. COHEN and NORBERT BERNDT The baculovirus/insect cell system compares favorably with bacterial, yeast, and mammalian expression systems in that it can be used to express eukaryotic genes as active nonfusion proteins that for the most part undergo posttranslational processing, are targeted to the correct organelle, and can be obtained in significant levels, even though they may be toxic to the cell) In this chapter we describe the application of this system for the high-level expression of protein phosphatase 1 (PP1), one of the major protein-serine/threonine phosphatases of eukaryotic cells: Although the expressed protein was mostly present as inactive, insoluble aggregates, complete reactivation could be achieved by a novel procedure that may be applicable to other protein-serine/threonine phosphatases. i V. A. L u c k o w and M. D. Summers, 2 p. Cohen, this volume [33].
METHODS IN ENZYMOLOGY,VOL. 201
Bio/Technology 6,
47 (1988).
Copyright © 1991by Academic Press, Inc. All rights of reproductionin any formreserved.
PROTEIN PHOSPHATASES
[34]
identity) than PP1 ( - 4 0 % identity). 6,46 It is clearly essential to study the enzymatic properties of these novel enzymes, either by isolating them or expressing the cDNA clones, in order to determine whether they can be classified as type 1 or type 2A, or whether their properties are so distinctive that additions to the current nomenclature are required.
[34] C l o n i n g o f P r o t e i n - S e r i n e / T h r e o n i n e
Phosphatases
By PATRICIA T. W. COHEN Four major types of protein-serine/threonine phosphatase (PP) catalytic subunit (PP1, PP2A, PP2B, and PP2C) have been identified in mammalian cells on the basis of their enzymatic properties.~ Over the past few years cDNAs encoding each of these enzymes have been cloned by constructing suitable oligonucleotides to peptide sequences derived from the purified phosphatases, and employing them in hybridization studies to isolate appropriate clones from bacteriophage cDNA libraries. Since these approaches have been used to clone many other proteins, they will be described fairly briefly in this chapter with emphasis on aspects of the methodology that the author has found particularly useful or are not documented clearly elsewhere. Subsequent library screening with the cDNAs encoding PP1 and PP2A led to the isolation of isoforms, homologs from different species, and cDNAs encoding putative protein phosphatases that had not been detected previously by the techniques of protein chemistry and enzymology. Procedures for detecting such novel enzymes are also discussed in this chapter, because additional phosphatases related to PP1 and PP2A are likely to be present in eukaryotic cells. Procedures for Cloning of PP1 and PP2A
Isolation of Peptides for Sequence Analysis PP1 and PP2A are low-abundance proteins composing 3000 Ci/ mmol) in a standard reaction mixture, 9 proceeds virtually to completion, and removal of unincorporated ATP is unnecessary.
Screening of Bacteriophage Libraries Libraries in bacteriophage are utilized in order that large numbers of cDNA clones ( 1 0 5 - 1 0 6 pfu) can be rapidly screened. Since peptide sequences of PP1 and PP2A had been obtained from the rabbit skeletal muscle enzymes, cDNA libraries are constructed in phage hgtl0 ~° from rabbit skeletal muscle and liver RNA using cDNA synthesized by the method of Gubler and Hoffman,ll or purchased from Clontech (Palo Alto, CA). Libraries are then screened by hybridization after in situ amplification of bacteriophage plaques. ~2All materials and procedures not described
6 y . Takahashi, K. Kato, Y. Hayashizaki, T. Wakabayashi, E. Ohtsuka, S. Matsuki, M. Ikehara, and K. Matsubara, Proc. Natl. Acad. Sci. U.S.A. 82, 1931 (1985). 7 F. H. Martin and M. M. Castro, Nucleic Acids Res. 13, 8927 (1985). 8 D. Guerini and C. Klee, Proc. Natl. Acad. Sci. U.S.A. 86, 9183 (1989). 9 R. B. Wallace and C. G. Miyada, this series, Vol. 152, p. 432. l0 T. V. Huynh, R. A. Young, and R. W. Davis, in " D N A Cloning: A Practical Approach" (D. M. Glover, ed.), Vol. 1, p. 49. IRL Press, Oxford, 1985. n U. Gubler and B. J. Hoffman, Gene 25, 263 (1983). ~2 S. L. C. Woo, this series, Vol. 68, p. 389.
[34]
CLONING OF PROTEIN-SERINE/THREONINE
PHOSPHATASES
401
in detail below can be found in Maniatis et al. 13The following modifications proved to be effective. 1. To encourage preservation of poorly growing clones, recombinant hgtl0 phage and host Escherichia coli C600 Hfl cells (Clontech) are plated on LB plates containing 10 mM MgSO4, since Mg2÷ helps stabilize phage heads. In addition, -85% identity), as are the isoforms of PP2A (>95% identity), virtually all the differences being confined to the N and C termini. In Drosophila, three isoforms of PP1 have been identified from their cDNAs. 3° Two of the Drosophila PP1 isoforms are very similar to mammalian P P l a , while the other would appear to be the homolog of mammalian PPlfl. An a-like PP1 (dis2), as well as a further isoform (sds21), are also present in Schizosaccharomyces p o m b e . 39 c D N A cloning of PP2B from mammalian brain has revealed two isoforms (PP2Ba and PP2B/3) which are the products of separate genes and show 82% overall identity. El Two further forms of PP2Ba 4° and three of PP2Bfl 8,41 can be generated by alternative splicing at the 3' end, and, in the case of one PP2B/3 form, additional splicing near the 5' end. 8 The structure of one isoform of PP2C (termed PP2Ca) has been determined by c D N A cloning, z2 PP2Ca and PP2C/3 have been purified from both rabbit skeletal muscle and rabbit liver and showed 80% amino acid sequence identity over the 62 residues where they could be compared directly.4Z Two Distinct Protein-Serine/Threonine Phosphatase Gene Families
The amino acid sequences deduced from the cDNAs of mammalian PP I and PP2A show 41% overall identity, indicating that they are members of the same gene family. The most divergent regions are the N and C termini, and the identity is 49% in the catalytic domain (Table I). The novel protein phosphatases PPX, 25 ppy,26 ppz25 and PPV, 25 and a yeast phosphatase, termed SIT4, 43 have similar domain structures to PP1 and PP2A and their identities to PP1 and PP2A are summarized in Table I. PPY and PPZ are more similar to PP1, while PPX, PPV, and SIT4 resemble PP2A more closely. PP2B is a member of the same gene family, but a more 39H. Ohkura, N. Kinoshita, S. Migatani, T. Toda, and M. Yanagida, Cell (Cambridge, Mass. ) 57, 997 (1989). 40R. L. Kincaid, R. Rathna Giri, S. Higuchi, J. Tamura, S. C. Dixon, C. A. Marietta, D. A. Amorese, and B. M. Martin, J. Biol. Chem. 265, 11312(1990). 41A. E. McPartlin, H. M. Barker, and P. T. W. Cohen, Biochim. Biophys. Acta, 1088, 308 (1991). 42C. H. McGowan, Ph.D. Thesis, University of Dundee (1988). 43K. T. Arndt, C. A. Styles, and G. R. Fink, Cell (Cambridge, Mass.) 56, 527 (1989).
406
PROTEIN PHOSPHATASES
.¢
2
¢q
o
e~
[34]
.'~.
oo
eq Z g]
"
go oq
< Z ¢) . .
0
"
go
o
Z go
.-t2. .1 e~
.
1 l>l :>1 ~1 I>l I>1 I~ I~ I¢I E-I E~ E-I e'l E'I l~
E ~ = o'l::
~m(
'W gle¢
~'r"
~
~
0
..
o
408
PROTEIN PHOSPHATASES
[35]
distantly related one. Its catalytic domain shows less similarity to PP1 and PP2A, and in addition it possesses two insertions in the catalytic domain as well as a long C-terminal extension that is involved in interaction with calmodulin. Bacteriophage h ORF221 is the shortest protein phosphatase known (221 amino acids) and the most divergent member of this family (Table 1). 36'37 Although the overall sequence identity is low (Table I), the Nterminal half of ORF221 shows 35% identity to PP1 or PP2A and includes the three highly conserved sections, corresponding to residues 63-67, 90-97, and 121-126 of P P l a (Fig. I). It is presumed that these regions are part of the active site. Oligonucleotides synthesized to these most highly conserved regions may prove useful for cloning protein phosphatases in this family that are highly divergent from PP1 and PP2A. This strategy has been successful for protein kinases. 44:5 PP2C, investigated both by peptide sequencing42 and cDNA cloning; 22 has no discernable sequence similarities to the PP1/PP2A/PP2B/hORF221 family. Thus it is a member of a quite distinct protein-serine/threonine phosphatase gene family. 44 S. K. Hanks and R. A. Lindberg, this series, Vol. 200, p. 525. 45 A. E. Wilks, this series, Vol. 200, p. 533.
[35] R e a c t i v a t i o n o f P r o t e i n P h o s p h a t a s e 1 E x p r e s s e d at High Levels from Recombinant Baculovirus
By PATRICIA T. W. COHEN and NORBERT BERNDT The baculovirus/insect cell system compares favorably with bacterial, yeast, and mammalian expression systems in that it can be used to express eukaryotic genes as active nonfusion proteins that for the most part undergo posttranslational processing, are targeted to the correct organelle, and can be obtained in significant levels, even though they may be toxic to the cell) In this chapter we describe the application of this system for the high-level expression of protein phosphatase 1 (PP1), one of the major protein-serine/threonine phosphatases of eukaryotic cells: Although the expressed protein was mostly present as inactive, insoluble aggregates, complete reactivation could be achieved by a novel procedure that may be applicable to other protein-serine/threonine phosphatases. i V. A. L u c k o w and M. D. Summers, 2 p. Cohen, this volume [33].
METHODS IN ENZYMOLOGY,VOL. 201
Bio/Technology 6,
47 (1988).
Copyright © 1991by Academic Press, Inc. All rights of reproductionin any formreserved.
