[49]
HETEROGENEITYOF PLC
511
min to an HPLC phenyl-5-PW column (7.5 × 75 mm) equilibrated with 20 mM HEPES, pH 7.0, 3 M KCI, 1 mM EGTA, and 0.1 mM DT. The proteins are eluted at a flow rate of 1 ml/min, by successive applications of (1) the equilibration buffer for 5 min, (2) a decreasing KCI gradient from 3 to 1.2 M for 10 min, and (3) a decreasing KCI gradient from 1.2 to 0 M for 25 min. Then the column is washed with a KCl-free buffer. Fractions of 1.0 ml are collected. PLC-T is eluted in a peak centered at 30 min. Peak fractions are pooled and washed with 50 mM Tris-HCl buffer, pH 7.6, containing 1 mM EGTA and 0.1 mM DTT. Step 14: Ion-Exchange Chromatography of PLC-T on a Mono-Q Column. The washed PLC-7 samples (5 ml) are applied at a flow rate of 1 ml/min to a Mono Q column (70 x 6 mm) equilibrated with 20 mM TrisHCI, pH 7.6, I mM EGTA, and 0.1 mM DTT. The proteins are eluted at a flow rate of 1.0 ml/min by successive applications of KCI gradients from 0 to 0.3 M for 20 min and from 0.3 to 1.0 M for 10 min. A major protein peak coinciding with PLC activity emerged at 20 min. Peak fractions (3 ml) are pooled, washed with 20 mM Tris-HC! buffer, pH 7.6, containing 0.1 mM DTT and 1 mM EGTA, concentrated to approximately 1 ml, separated into aliquots, and stored at - 2 0 °. This procedure yields about 4 mg of PLC-T (>95% pure).
[49] P r o p e r t i e s o f P h o s p h o l i p a s e C I s o z y m e s
By TADAOMI TAKENAWA, YOSHIMI HOMMA, and YASUFUM1 EMORI Introduction
Phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) has been proposed to be a crucial step in the cellular response to calcium-mobilizing hormones and neurotransmitters. 1-3 Numerous attempts to isolate PLC from various tissues have been made. As a result, many kinds of PLC have been isolated and characterized from various mammalian tissues. We have also purified a PLC from rat liver. T M I M. J. Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 2 y. Nishizuka, Science 225, 1365 (1984). 3 p. W. Majerus, T. M. Connolly, H. Dechmyn, T. S. Ross, T. E. Bross, H. lshii, V. S. Bansal, and D. B. Willson, Science 234, 1519 (1986). 4 T. Takenawa and Y. Nagai, J. Biol. Chem. 256, 6769 (1981). 5 S. L. Hofmann and P. W. Majerus, J. Biol. Chem. 257, 6461 (1982). 6 H. Hakata, J. Kobayashi, and G. Kosaki, J. Biochem. (Tokyo) 92, 929 (1982).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
512
PHOSPHOLIPASE C
[49]
Phospholipase C in R a t Brain Ryu e t al. 7 isolated from bovine brain three types of P L C , having molecular weights of 150K, 145K, and 85K, respectively, when measured by sodium dodecyl sulfate (SDS)-gel electrophoresis. M o r e o v e r , the three P L C s are immunologically distinct as evidenced by the fact that monoclonal antibodies directed against each e n z y m e do not cross-react, demonstrating the presence o f P L C i s o z y m e s in brain. We also isolated from rat brain two types of P L C with molecular weights for both of 85K, although their immunological reactivity was different. 8 One was found to be same as the P L C (85K) reported by Ryu e t al. previously. Therefore, at least four types of P L C m a y exist in rat brain. According to R h e e ' s classification, ~5 these P L C s are divided to types fl, 3', 8, and e, respectively. We purified these types of P L C from rat brain and examined their properties J6 (summarized in Table I). PLC-fl was purified f r o m rat brain m e m b r a n e fractions, and PLC-y, PLC-8, and P L C - e were from rat brain cytosol fractions. The molecular weight of PLC-fl was found to be 150K. The contribution of PLC-fl to total P L C activity was found to be about 50%, suggesting that PLC-fl shares the largest activity in rat brain. PLC-fl required fairly high Ca 2+ concentrations (10/xM) for hydrolysis of PIP2, but a m u c h higher concentration of Ca 2÷ was needed to hydrolyze PI (1 m M or more). PLC-~/, which corresponds to the 145K P L C purified by Ryu e t al. from bovine brain, showed similar properties to those of PLC-/3 on the basis of substrate specificity and Ca 2+ dependence. P L C - y accounted for around 20% of the total P L C activity. PLC-8 and P L C - e were purified 2810-fold and 4010fold, respectively, to homogeneity.8'J6 The molecular weight of these P L C s was estimated to be 85K. P L C - a accounted for about 20-25% of the total activity in rat brain, but the activity of PLC-e was less than 5%. Like
7 S. H. Ryu, P. G. Suh, K. S. Cho, K. Y. Lee, and S. G. Rhee, Proc. Natl. Acad. Sci. U.S.A. 84, 6649 (1987). 8 y. Homma, J. lmaki, O. Nakanishi, and T. Takenawa, J. Biol. Chem. 263, 6592 (1988). 90. Nakanishi, Y. Homma, H. Kawasaki, Y. Emori, K. Suzuki, and T. Takenawa, Biochem. J. 256, 453 (1988). l0 y. Banno, Y. Yada, and Y. Nozawa, J. Biol. Chem. 263, 11459 (1988). ii C. F. Bennett and S. T. Crooke, J. Biol. Chem. 262, 13789 (1987). 12M. Katan and P. J. Parker, Eur. J. Biochem. 168, 413 (1987). 13M. J. Rebecchi and O. M. Rosen, J. Biol. Chem. 262, 12526 (1987). 14T. Fukui, R. J. Lutz, and J. M. Lowenstein, J. Biol. Chem. 263, 17730 (1988). 15S. G. Rhee, P.-G. Suh, S.-H. Ryu, and S. Y. Lee, Science 244, 546 (1989). t6 T. Takenawa, Y, Homma, and O. Nakanishi, in "Physiology and Pharmacology of Transmembrane Signalling" (T. Segawa, M. Endo, M. Ui, and K. Kurihara, eds.), p. 207. Elsevier, Amsterdam, 1989.
[49]
HETEROGENEITY OF P L C
513
TABLE I PROPERTIES OF PHOSPHOLIPASE C ISOZYMES IN RAT BRAIN a T y p e of
Optimal Ca 2+
Substrate
Vmax (/~mol/min/ng)
Km
PLC
(p,M)
(M)
fl
PIP2 PIP PI PIP 2 PIP Pl PIP 2 PIP PI PIP 2 PIP PI
25.3 12.9 20.1 16.2 12.3 18.3 15.3 8.4 19.2 12.9 6.5 i.3
120 -90 140 -100 200 125 135 130 80 >200
10 -5 10-5-10 -4 > 1 0 -3 10 -5 10-5-10 -4 > 1 0 -3 5 × 10 -5 10 -4 >10 -3 10 -6 5 × 10 -6 >10 -3
y
8
e
a PIP2, Phosphatidylinositol 4,5-biphosphate; PIP, phosphatidylinositol 4-phosphate; PI,
phosphatidylinositol.
PLC-fl and PLC-% these enzyme required high C a 2+ concentrations to hydrolyze PI. PLC-e especially needed much higher Ca 2÷ concentrations and did not show any activity for PI hydrolyis at a Ca 2÷ level lower than 1 mM. When PIP2 was used as substrate, PLC activity could be detected at lower Ca 2÷ concentrations. Optimal Ca 2÷ concentrations for PLC-fl, PLC-3,, and PLC-8 were around 10/xM, whereas that for PLC-e was found to be very low (1/zM or less). However, K m and Vmaxvalues were almost identical among PLC isozymes when PIP2 was used as substrate. Recently, cDNA clones corresponding to PLC-ct, -/3, -7, and -~i have also been isolated. 17-20Three PLCs (fl, % and 8) had two conserved regions considered to be catalytic domains for PLC activity in common. However, another PLC (a) had a totally different amino acid sequence showing similarity to thioredoxin of Escherichia coli.~7 These results suggest that there are several genes encoding PLCs having completely different structures. J7 C. F. Bennett, J. M. Malcareck, A. Varrichio, and S. T. Crook, Nature (London) 334, 268 (1988). 18 M. Katan, R. W. Kriz, N. Totty, R. Philip, E. Meldrum, R. A. Aldalpe, J. L. Knopf, and P. J. Parker, Cell (Cambridge, Mass.) 54, 171 (1988). 19 M. L. Stalh, C. R. Ferenz, K. L. Kellehe, R. W. Kriz, and J. L. Knopf, Nature (London) 332, 209 (1988). 2o p. G. Suh, S. H. Ryu, K. H. Moon, H. W. Suh, and S. G. Rhee, Cell (Cambridge, Mass.) 54, 161 (1988).
514
PHOSPHOLIPASE C
I
x
II
¥
I
x
I-t
z
.,c,.-!
x
H
PLC "Y2 N •~
X
H
"
[49] c
I
l--c
PI
¥
J'-"l
Y F°
tc z
100 a.a.
FIG. 1. Schematic structure of four isozymes of PLC. Homologous domains common to PLC-fl, -3q, -"/2,and -8 are represented by X and Y, and the src-related domain is represented by Z. a.a., Amino acid. Phospholipase C Y2 In addition to e D N A clones for various P L C s (a, fl, y, and 8) we have isolated a e D N A clone encoded a new type of P L C from a rat muscle e D N A library that contains a single open reading f l a m e encoding 1265 amino acid residues. 17 The deduced amino acid sequence differs from those of P L C - a , -fl, -% and -6, though it contains two homologous regions c o m m o n to PLC-fl, -Y, and -8 (Fig. 1). These two regions were previously designated as domains X and Y by Rhee e t al.~5 Like PLC-y, this P L C could be structually divided into three domains (X, Y, and Z). 17 The first and third domains (domains X and Y) are c o m m o n to PLC-fl, -y, and -8. The second domain (domain Z), also found in PLC-y, is related to the Nterminal regulatory domains of oncogenes in the s r c family. Therefore, the structure of this P L C is most similar to that of PLC-y, and 50.2% of the amino acid residues were identical. As reported previously,~5 domain Z contains three subdomains, A, B - e l , and B-C2, which are also present in the new P L C (Fig. 2). Although the functions of the homologous sequences are not clear, a regulatory role of domain Z on P L C activity is suggested because of homology with the regulatory portion of s r c family tyrosine kinases, c r k , and the GTP-activating protein of r a s p2119"21-23 Amino acid residues of domain Z in the new P L C are 60% identical to those of P L C - y . Thus, this P L C should be n a m e d PLC-yz, with the P L C - y previously reported designated as P L C - y l . _,l y. Emori, Y. Homma, H. Sorimachi, H. Kawasaki, O. Nakanishi, K. Suzuki, and T. Takenawa, J. Biol. Chem. 261, 21886, (1989). 22 B. J. Mayer, M. Hamaguchi, and H. Hanafusa, Nature (London) 332, 272 (1988). -,3 U. S. Vogel, R. A. Dixon, M. D. Schaber, R. F. Diehl, M. S. Marshall, E. M. Scolnick, 1. S. Sigal, and J. B. Gibbs, Nature (London) 335, 90 (1988).
[49]
HETEROGENEITYOF PLC
515
Z Region
'~=
Y2
• ,~
''
" ....
I "'-c' P - I
.-c.
i
F~,
.-
I ,'-cl !--I
,-c.
I
~--I---c
.-c
~
.
g.,
I*
.'
oA,
~
" ....
I
I
"-c'
.-c
I
c
.c
l--IKln..,
l°-c"
c
I
"'-°
100 s . s . I
1
FIG. 2. Schematic representation of relevant regions of PLC-3q and PLC-y2, oncogenes
(src and crk), and the GTP-activating protein (GAP). Regions that exhibit similarity are represented by A and B-C. In cases where two homologous regions were observed, they are designated B-CI and B-C2.
We have established an expression system for PLC in E. coli and determined the essential region for PLC activity. Analysis revealed that even small deletions in domain X result in a complete loss of PLC activity and that deletion of the N-terminal 220 residues, which are not contained in previously described homologous regions, also cause the loss of activity. In addition, a precise homology search showed that the X and Y regions are longer than previously reported. Thus, domain X contains about 400 amino acid residues (from 50 to 450 of PLC-y2) and domain Y contains about 270 residues (from 930 to 1200) (Fig. 1). These domains are essential for enzymatic activity. Therefore, PLC-8 is considered to have the minimal structure for PLC, since a region not included in domains X and Y is very short in PLC-& On the other hand, PLC-y~ and -3'2 contain a long intervening region (domain Z) between the X and Y domains. PLC-/3 has a unique long C-terminal sequence rich in basic amino acid residues and a unique sequence with a cluster of Glu residues between the X and Y domains. These regions may play important roles in the regulation of PLC activity through the interaction with other molecules. However, even after
516
PHOSPHOLIPASE C
[49]
ant/-PLC- ~ 2 31333537
39
4/
4345
0.15
3 D
2
.g
0.4
0.1o E o
0.2
0 0
I
I
50
100
0.05
0
Fraction anti-PLC- ~ = t5
0.3
~" 1.0 J 5
0.4 02
o.s
HETEROGENEITYOF PLC
511
min to an HPLC phenyl-5-PW column (7.5 × 75 mm) equilibrated with 20 mM HEPES, pH 7.0, 3 M KCI, 1 mM EGTA, and 0.1 mM DT. The proteins are eluted at a flow rate of 1 ml/min, by successive applications of (1) the equilibration buffer for 5 min, (2) a decreasing KCI gradient from 3 to 1.2 M for 10 min, and (3) a decreasing KCI gradient from 1.2 to 0 M for 25 min. Then the column is washed with a KCl-free buffer. Fractions of 1.0 ml are collected. PLC-T is eluted in a peak centered at 30 min. Peak fractions are pooled and washed with 50 mM Tris-HCl buffer, pH 7.6, containing 1 mM EGTA and 0.1 mM DTT. Step 14: Ion-Exchange Chromatography of PLC-T on a Mono-Q Column. The washed PLC-7 samples (5 ml) are applied at a flow rate of 1 ml/min to a Mono Q column (70 x 6 mm) equilibrated with 20 mM TrisHCI, pH 7.6, I mM EGTA, and 0.1 mM DTT. The proteins are eluted at a flow rate of 1.0 ml/min by successive applications of KCI gradients from 0 to 0.3 M for 20 min and from 0.3 to 1.0 M for 10 min. A major protein peak coinciding with PLC activity emerged at 20 min. Peak fractions (3 ml) are pooled, washed with 20 mM Tris-HC! buffer, pH 7.6, containing 0.1 mM DTT and 1 mM EGTA, concentrated to approximately 1 ml, separated into aliquots, and stored at - 2 0 °. This procedure yields about 4 mg of PLC-T (>95% pure).
[49] P r o p e r t i e s o f P h o s p h o l i p a s e C I s o z y m e s
By TADAOMI TAKENAWA, YOSHIMI HOMMA, and YASUFUM1 EMORI Introduction
Phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) has been proposed to be a crucial step in the cellular response to calcium-mobilizing hormones and neurotransmitters. 1-3 Numerous attempts to isolate PLC from various tissues have been made. As a result, many kinds of PLC have been isolated and characterized from various mammalian tissues. We have also purified a PLC from rat liver. T M I M. J. Berridge and R. F. Irvine, Nature (London) 312, 315 (1984). 2 y. Nishizuka, Science 225, 1365 (1984). 3 p. W. Majerus, T. M. Connolly, H. Dechmyn, T. S. Ross, T. E. Bross, H. lshii, V. S. Bansal, and D. B. Willson, Science 234, 1519 (1986). 4 T. Takenawa and Y. Nagai, J. Biol. Chem. 256, 6769 (1981). 5 S. L. Hofmann and P. W. Majerus, J. Biol. Chem. 257, 6461 (1982). 6 H. Hakata, J. Kobayashi, and G. Kosaki, J. Biochem. (Tokyo) 92, 929 (1982).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
512
PHOSPHOLIPASE C
[49]
Phospholipase C in R a t Brain Ryu e t al. 7 isolated from bovine brain three types of P L C , having molecular weights of 150K, 145K, and 85K, respectively, when measured by sodium dodecyl sulfate (SDS)-gel electrophoresis. M o r e o v e r , the three P L C s are immunologically distinct as evidenced by the fact that monoclonal antibodies directed against each e n z y m e do not cross-react, demonstrating the presence o f P L C i s o z y m e s in brain. We also isolated from rat brain two types of P L C with molecular weights for both of 85K, although their immunological reactivity was different. 8 One was found to be same as the P L C (85K) reported by Ryu e t al. previously. Therefore, at least four types of P L C m a y exist in rat brain. According to R h e e ' s classification, ~5 these P L C s are divided to types fl, 3', 8, and e, respectively. We purified these types of P L C from rat brain and examined their properties J6 (summarized in Table I). PLC-fl was purified f r o m rat brain m e m b r a n e fractions, and PLC-y, PLC-8, and P L C - e were from rat brain cytosol fractions. The molecular weight of PLC-fl was found to be 150K. The contribution of PLC-fl to total P L C activity was found to be about 50%, suggesting that PLC-fl shares the largest activity in rat brain. PLC-fl required fairly high Ca 2+ concentrations (10/xM) for hydrolysis of PIP2, but a m u c h higher concentration of Ca 2÷ was needed to hydrolyze PI (1 m M or more). PLC-~/, which corresponds to the 145K P L C purified by Ryu e t al. from bovine brain, showed similar properties to those of PLC-/3 on the basis of substrate specificity and Ca 2+ dependence. P L C - y accounted for around 20% of the total P L C activity. PLC-8 and P L C - e were purified 2810-fold and 4010fold, respectively, to homogeneity.8'J6 The molecular weight of these P L C s was estimated to be 85K. P L C - a accounted for about 20-25% of the total activity in rat brain, but the activity of PLC-e was less than 5%. Like
7 S. H. Ryu, P. G. Suh, K. S. Cho, K. Y. Lee, and S. G. Rhee, Proc. Natl. Acad. Sci. U.S.A. 84, 6649 (1987). 8 y. Homma, J. lmaki, O. Nakanishi, and T. Takenawa, J. Biol. Chem. 263, 6592 (1988). 90. Nakanishi, Y. Homma, H. Kawasaki, Y. Emori, K. Suzuki, and T. Takenawa, Biochem. J. 256, 453 (1988). l0 y. Banno, Y. Yada, and Y. Nozawa, J. Biol. Chem. 263, 11459 (1988). ii C. F. Bennett and S. T. Crooke, J. Biol. Chem. 262, 13789 (1987). 12M. Katan and P. J. Parker, Eur. J. Biochem. 168, 413 (1987). 13M. J. Rebecchi and O. M. Rosen, J. Biol. Chem. 262, 12526 (1987). 14T. Fukui, R. J. Lutz, and J. M. Lowenstein, J. Biol. Chem. 263, 17730 (1988). 15S. G. Rhee, P.-G. Suh, S.-H. Ryu, and S. Y. Lee, Science 244, 546 (1989). t6 T. Takenawa, Y, Homma, and O. Nakanishi, in "Physiology and Pharmacology of Transmembrane Signalling" (T. Segawa, M. Endo, M. Ui, and K. Kurihara, eds.), p. 207. Elsevier, Amsterdam, 1989.
[49]
HETEROGENEITY OF P L C
513
TABLE I PROPERTIES OF PHOSPHOLIPASE C ISOZYMES IN RAT BRAIN a T y p e of
Optimal Ca 2+
Substrate
Vmax (/~mol/min/ng)
Km
PLC
(p,M)
(M)
fl
PIP2 PIP PI PIP 2 PIP Pl PIP 2 PIP PI PIP 2 PIP PI
25.3 12.9 20.1 16.2 12.3 18.3 15.3 8.4 19.2 12.9 6.5 i.3
120 -90 140 -100 200 125 135 130 80 >200
10 -5 10-5-10 -4 > 1 0 -3 10 -5 10-5-10 -4 > 1 0 -3 5 × 10 -5 10 -4 >10 -3 10 -6 5 × 10 -6 >10 -3
y
8
e
a PIP2, Phosphatidylinositol 4,5-biphosphate; PIP, phosphatidylinositol 4-phosphate; PI,
phosphatidylinositol.
PLC-fl and PLC-% these enzyme required high C a 2+ concentrations to hydrolyze PI. PLC-e especially needed much higher Ca 2÷ concentrations and did not show any activity for PI hydrolyis at a Ca 2÷ level lower than 1 mM. When PIP2 was used as substrate, PLC activity could be detected at lower Ca 2÷ concentrations. Optimal Ca 2÷ concentrations for PLC-fl, PLC-3,, and PLC-8 were around 10/xM, whereas that for PLC-e was found to be very low (1/zM or less). However, K m and Vmaxvalues were almost identical among PLC isozymes when PIP2 was used as substrate. Recently, cDNA clones corresponding to PLC-ct, -/3, -7, and -~i have also been isolated. 17-20Three PLCs (fl, % and 8) had two conserved regions considered to be catalytic domains for PLC activity in common. However, another PLC (a) had a totally different amino acid sequence showing similarity to thioredoxin of Escherichia coli.~7 These results suggest that there are several genes encoding PLCs having completely different structures. J7 C. F. Bennett, J. M. Malcareck, A. Varrichio, and S. T. Crook, Nature (London) 334, 268 (1988). 18 M. Katan, R. W. Kriz, N. Totty, R. Philip, E. Meldrum, R. A. Aldalpe, J. L. Knopf, and P. J. Parker, Cell (Cambridge, Mass.) 54, 171 (1988). 19 M. L. Stalh, C. R. Ferenz, K. L. Kellehe, R. W. Kriz, and J. L. Knopf, Nature (London) 332, 209 (1988). 2o p. G. Suh, S. H. Ryu, K. H. Moon, H. W. Suh, and S. G. Rhee, Cell (Cambridge, Mass.) 54, 161 (1988).
514
PHOSPHOLIPASE C
I
x
II
¥
I
x
I-t
z
.,c,.-!
x
H
PLC "Y2 N •~
X
H
"
[49] c
I
l--c
PI
¥
J'-"l
Y F°
tc z
100 a.a.
FIG. 1. Schematic structure of four isozymes of PLC. Homologous domains common to PLC-fl, -3q, -"/2,and -8 are represented by X and Y, and the src-related domain is represented by Z. a.a., Amino acid. Phospholipase C Y2 In addition to e D N A clones for various P L C s (a, fl, y, and 8) we have isolated a e D N A clone encoded a new type of P L C from a rat muscle e D N A library that contains a single open reading f l a m e encoding 1265 amino acid residues. 17 The deduced amino acid sequence differs from those of P L C - a , -fl, -% and -6, though it contains two homologous regions c o m m o n to PLC-fl, -Y, and -8 (Fig. 1). These two regions were previously designated as domains X and Y by Rhee e t al.~5 Like PLC-y, this P L C could be structually divided into three domains (X, Y, and Z). 17 The first and third domains (domains X and Y) are c o m m o n to PLC-fl, -y, and -8. The second domain (domain Z), also found in PLC-y, is related to the Nterminal regulatory domains of oncogenes in the s r c family. Therefore, the structure of this P L C is most similar to that of PLC-y, and 50.2% of the amino acid residues were identical. As reported previously,~5 domain Z contains three subdomains, A, B - e l , and B-C2, which are also present in the new P L C (Fig. 2). Although the functions of the homologous sequences are not clear, a regulatory role of domain Z on P L C activity is suggested because of homology with the regulatory portion of s r c family tyrosine kinases, c r k , and the GTP-activating protein of r a s p2119"21-23 Amino acid residues of domain Z in the new P L C are 60% identical to those of P L C - y . Thus, this P L C should be n a m e d PLC-yz, with the P L C - y previously reported designated as P L C - y l . _,l y. Emori, Y. Homma, H. Sorimachi, H. Kawasaki, O. Nakanishi, K. Suzuki, and T. Takenawa, J. Biol. Chem. 261, 21886, (1989). 22 B. J. Mayer, M. Hamaguchi, and H. Hanafusa, Nature (London) 332, 272 (1988). -,3 U. S. Vogel, R. A. Dixon, M. D. Schaber, R. F. Diehl, M. S. Marshall, E. M. Scolnick, 1. S. Sigal, and J. B. Gibbs, Nature (London) 335, 90 (1988).
[49]
HETEROGENEITYOF PLC
515
Z Region
'~=
Y2
• ,~
''
" ....
I "'-c' P - I
.-c.
i
F~,
.-
I ,'-cl !--I
,-c.
I
~--I---c
.-c
~
.
g.,
I*
.'
oA,
~
" ....
I
I
"-c'
.-c
I
c
.c
l--IKln..,
l°-c"
c
I
"'-°
100 s . s . I
1
FIG. 2. Schematic representation of relevant regions of PLC-3q and PLC-y2, oncogenes
(src and crk), and the GTP-activating protein (GAP). Regions that exhibit similarity are represented by A and B-C. In cases where two homologous regions were observed, they are designated B-CI and B-C2.
We have established an expression system for PLC in E. coli and determined the essential region for PLC activity. Analysis revealed that even small deletions in domain X result in a complete loss of PLC activity and that deletion of the N-terminal 220 residues, which are not contained in previously described homologous regions, also cause the loss of activity. In addition, a precise homology search showed that the X and Y regions are longer than previously reported. Thus, domain X contains about 400 amino acid residues (from 50 to 450 of PLC-y2) and domain Y contains about 270 residues (from 930 to 1200) (Fig. 1). These domains are essential for enzymatic activity. Therefore, PLC-8 is considered to have the minimal structure for PLC, since a region not included in domains X and Y is very short in PLC-& On the other hand, PLC-y~ and -3'2 contain a long intervening region (domain Z) between the X and Y domains. PLC-/3 has a unique long C-terminal sequence rich in basic amino acid residues and a unique sequence with a cluster of Glu residues between the X and Y domains. These regions may play important roles in the regulation of PLC activity through the interaction with other molecules. However, even after
516
PHOSPHOLIPASE C
[49]
ant/-PLC- ~ 2 31333537
39
4/
4345
0.15
3 D
2
.g
0.4
0.1o E o
0.2
0 0
I
I
50
100
0.05
0
Fraction anti-PLC- ~ = t5
0.3
~" 1.0 J 5
0.4 02
o.s
