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Proteolytic fragment of protein kinase C (kinase M) phosphorylates in vitro phosphatidylinositol-4-phosphate
Vol.
176,
No.
May
15,
1991
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
PROTEOLYTIC PHOSPHORYLATES
FRAGMKNT OF PROTEIN KINASE C (KINASE IN VITRO PHOSPHATIDYLINOSITOL-&PHOSPHATE
0. K. Tusupov,
S. E. Severin,*+
1007-1013
M)
and V. I. Shvets
Fine Chemical Technology, M.V. Lomonosov Institute of * Moscow, and Institute of Diabetes, USSR Academy of Medical Science,117036 Moscow, USSR Received
March
15,
1991
Summary: Limited tryptic proteolysis of homogeneous protein kinase C induces the formation of a catalytically active unlike native PK C fragment of 50 kDa (kinase M) which, the ability to phosphorylate PIP. Both ATP and acquires GTP were found to be capable of serving as phosphate donors in this process. Incubation of purified kinase M with a preparation of rat brain membrane fraction enha;;;d the level of phosphorylation of PIP in the presence in the absence of exogenous PIP. A scheme of the interrelationship of phosphoinositide metabolism and the proteolytic processingof protein kinase C is proposed. 01991
Ca2+ ,phospholipid-dependent protein kinase (PK C) is essential for regulatory processes of realizing the cell metabolism resulting from receptor-induced hydrolysis of phosphoinositides (1). One of the products of the phosphoinositide turnover 1,2-diacyl-sn-glycerol (DAG) stimulates protein kinase C, whereas the other, inositol1,4,5trisphosphate (IP3), mobilizes the intracellular Ca 2+. Furthermore, a proteolytically activated form of PK C (protein kinase M (PK M)) that widely occurs in many organs PK C proteolysis and tissues was shown to exist (3). action of calpain after the apparently occurs under the
+To whom correspondence and reprint requests should be addressed at Research Center of Molecular Diagnostics, Simpheropolsky Rlvd., S, 113149 Moscow, USSR. Abbreviations: PK C,protein kinase C;PIP,phosphatidylinositol-I-phosphat; phosphatidylinositol-4,5-bisphosphate; IP3, PIPzr PK M,protein kinase M; TPCK,L-linositol trisphosphate; -chlor-3-tosylamido-4-phenyl-2-butanon. 0006-291X/91
1007
$1.50
Copyight 0 1991 by Accrdernic Press, Inc. All rights of reproduction in any form reserved.
Vol.
176,
No.
3, 1991
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AND
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enzyme the membrane, activation and translocation to though physiological role of PK M remains unclear the (4,5).Treatment of intact cells with phospholipase C, phorbol esters, chemoattractants, i.e., agents that induce PK C translocation, was shown kinase M accumulation to stimulate (6-9). At the same time, another effect of the above subwas noted, namely the enhancement of stances on the cells lipids (10-12). Our phosphorylation of inositol-containing experimental data on isolation and analysis of phosphatidilinositol-4-phosphate kinase from human brain (unpublished result) and comparison of the enzyme properties with those of the catalytic fragment of PK C (PK M) enabled us to suggest that the latter can perform the function of the PIP kinase. and a hypothesis Evidence in support of the advanced suggestion in the about involvement of proteolytic PK C degradation and regulation of the cell responses are transduction offered below. MATERIALS
AND
Rat
METHODS
brain, line Wistar was used as the source for isolating protein kinase C. [Y-~~PIATP (3000 Ci/mmol) and [Y-~~PIGTP (3000 Ci/mmol) were purchased from Amercham. PIP and PIP2 were obtained as described in (13). All the rest of the reagents were from Sigma and Serva. PK C purification. The enzyme was purified by the ---------------method detailed in (14) or (15). In all cases the preparation was judging by the data homogeneous, of SDS electrophoresis in the system of Laemmli (16), and was a mixture of known PK C isoforms. PK C assay . The activity was assayed as described in (14) -w-w----with slight modifications in the reaction mixture (100 ul) containing 50 mM Tris-HCI buffer pH 7.5, 5 mM MgCI 2 1 mM DTT, 20 ug histone Hl, 0.6 mM CaC12, 50 uM ATP, [T-~~P]ATP (1 uCi), 2 ug PS and 0,2 ug dioleoylglycerol. The lipids were mixed and added into the reaction mixture in the form of sonicated suspension. The basal activity ( PK M activity ) was measured in the presence of 0.7 mM EGTA insteadof dioleoylglycerol, PS and CaC$ !CCyptic of PK C. Trypsin (64 U/mg) was treated ---- proteolysis ---a-----------with loo-fold excess of TPCK for inhibiting chymotrypsin admixtures in the 50 mM Tris-HCI buffer, pH 7.5, containing 0.25 mM EGTA, 0.25 mM EDTA, 0,6 mM CaC12, and purified by gel filtration on PD-10 columm (Pharmacia) in the same buffer, 0.1 mg/ml of the PK C preparation in the buffer of 20 mM Tris-HCI pH 7.5, 0.5 EGTA, 0.5 mM EDTA, 1mM DTT, 0.2 mM NaCI were incubated at 37 Y with TPCK-trypsin at the enzyme-substrate ratio of 1:50 for different time intervals.The reaction was terminated with the lofold excess ( over the trypsin amount ) of soy-bean trypsin inhibitor. The resultant preparation was at once used in experiments on histone and PIP phosphorylation. 1008
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3, 1991
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and thin-layer -____________ chromatography---of PIP~~k&z!se-------assay ----------------phosphoinositides. Phosphorylation was carried out in the --- -----------reaction mixture (100 ul) containing 50 mM Tris-HCI pH 7.5, 0.25 mM EDTA, 0.25 mM EGTA, 1 mM DTT, 0.1 M NaCI, 20 mM MgCIZ O,Ol% Triton X-100, 10 mM PIP and 50uM ATP [Y-~~P]ATP (2uCi) .The reaction was initiated by the addition of ATP and, following incubation for 5 min at room temperature (25 4, terminated through introducing into the reaction mixture of 0.3 ml of chloroform, 0.5 ml of the metanol-1N HCI (1:l) solution and 50 ~1 (10 )1g) of cold PIP2 in the chloroform-methanol (2:l) solution. After vigorous stirring, the tubes were centrifuged at 5000 rpm for 5 min for phase separation, whereupon the upper water phase was removed and the lower one was evaporated in a Speedvac concentrator. The evaporated lipids were dissolved in chloroform-methanol (2:1), applied to the plates with silica gel impregnated with 1% potassium oxalate solution, and chromatographed in the n-propanol-4N ammonia (2:l) system. Following autoradiography, the spots corresponding to the position of PIP2 standard were collected from the plate into vials, supplemented with 1 ml of the methanol-1N HCI (1:l) mixture, 9 ml of scintillation cocktail was added and radioactivity was measured in a liquid scintillation spectrometer. RESULTS
AND
DISCUSSION
Fig.1 shows the results of the in vitro experiment with a PK C preparation, which involved the following steps. Highly purified PK C was subjected to limited proteolysis with trypsin, resulting in the formation of a catalytic and a regulatory domain. The process has been described in the literature many times and the products of tryptic proteolysis have been characteAfter the reaction termination with a rized in details (16-18). trypsin inhibitor, optimal conditions were maintained in the system for manifestation of PIP kinase activity (19-21), i.e., Ca2+ was increased. Thereafter ions were bound and Mg2+ concentration the PIP suspension was added and the phosphorylation reaction was was run in painitiated by introducing ATP. The same procedure rallel for native PK C as well. As clear from Fig.lA, native PK C is incapable of phosphorylating PIP, whereas the trypsin-treated enzyme acquires such capacity. Moreover, the maximal level of is achieved as early as within 3 32P i incorporation into PIP 2 min. The following decrease in the label incorporation is well explained by the known fact of trypsin-effected PK C cleavage into smaller fragments that lose the enzymic activity. In a para3s i into histone Hl llel experiment (Fig.lB), incorporation of of native and trypsin-treated PK C was measured. The experimental results indicate that in the result of tryptic proteolysis, Ca2+ ,phospholipid-dependent activity of histone phosphorylation 1009
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-
PL 3717
-
354%
-
t
72%
207%
;1 100%
1
2
3
kl 45%
4
5
33
L
2
3
into PIP (A) and histone Fiaure l."- P incorporation H1 (B) 1, protein kinase C forms.A: Purified frown [y- '-P]ATP by different rat brain PK C (5yg) was incubated in conditions for PIP-kinase assay as described in Materials and Methods; control without enzyme (lane l), native enzyme (lane Z),enzyme incubated with 5 respectively). B: PK C trypsin lmin, 3min, 5min (lane 3, 4, (5pg) was incubated in conditions for PK C assay as described in Materials and Methods; native enzyme (lane 1), enzyme incubated with trypsin lmin, 3min (lane 2,3 respectively).
and the basal activity ( kinase M activity ) accordingly increases (Fig.lB). Since PK C is known to be incapable of using GTP as a phosphate donor, while PIP-kinase has been shown to the possess this capacity (19,21), we performed an experiment similar to the above described, except that ATP was substituted for GTP. The experimental results (Fig.2) evidence that the native PK C in the presence of GTP has no potency in phosphorylating PIP, whereas cleavage with trypsin promotes the emergence of this function (Fig.ZA). Meanwhile, neither the native enzyme nor the trypsintreated one can phosphorylate histone, once GTP is used (Fig.2B). The above findings allow a conclusion that the catalytic PK C fragment (kinase M) is able to perform the function of PIPkinase. As the process of phosphoinositide interconversion occurs in the plasmatic membranes of cells, we analysed the effect of exogenous kinase M on PIP phosphorylation in the crude membrane fraction formed following centrifugation of rat brain homogenate at 105,000 g. The data of Fig.3 demonstrate that incubation of decreases
1010
Vol.
176,
No.
3, 1991
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AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
182%
UL i
1
1f
-
2
4
1
2
3
Fiqure 2.32 P incorporation into PIP (A) and histone Hl (B) from protein kinase C forms.A: Purified rat [Y-32 PIGTP by different brain PK C (51J.g) was incubated in conditions for PIP-kinase assay as described in Materials and Methods; control without enzyme (lane l), native enzyme (lane 2),enzyme incubated with trypsin lmin, 3min (lane 3,4 respectively). B: PK C (5fig) was incubated in conditions for PK C assay as described in Materials and Methods: native enzyme (lane l), enzyme incubated with trypsin lmin, 3min (lane 2,3 respectively).
the membrane with a kinase M preparation, obtained after cleavage of homogeneous PK C (15) and subsequent purification of the catalytic fragment by DEAE-cellulose chromatography (16), enhances the phosphate incorporation from ATP into PIP2 both in the presence and in the absence of exogenous PIP.
2421
I
PIP
+
+ +
PK n
Piqure 3. Enhancement of 32P incorporation phosphatidylinositol-4-phosphat from crude purified kinase M.
1011
+
from [Y-~~PIATP membrane fraction
into by
Vol.
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No.
3, 1991
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Fioure 4. Proposed scheme interrelationship PK C and phosphoinosiPIP2(step l), turnover: receptor-dependent hydrolys tide 2), protein kinase C and enhancement intracellular Ca ++ (step C and calpain translocation (step 3), proteolysis protein kinase phosphorylation of phosphatidylinositol-4-phosphate by kinase M.
Proceeding from the data of this study as well as known facts that both PK C and calpain in the presence of Ca2+ ions are mainly located on the membrane (22) and, moreover, calpain can be associated with PK C (23), we propose the following hypothetical scheme of interrelationship between PK C and PIP-kinase. Activation of phosphatidylinositol-specific phospholipase C via a corresponding cell receptor leads to a sharp decrease in the PIP2 2t concentration and elevation of Ca , which, in turn, causes PK C translocation to the membrane structures and activation of phosphorylation. A simultaneous activation of calpain will take the latter, while cleaving native PK C, increases the place, kinase M level in the near-membrane region. The enzyme will promote restoration of PIP2 In other words, the proteolytic fragment of PK C acts as a feedback in the case of receptordependent PIP2 degradation (Fig.4). REFERENCES 1. Nishizuka,Y.(1988) Nature 334,661-665 2. Streb,M.D.,Irvine,R.F.,Berridge,M.J.,Schulz,J.(l983) Nature 306, 67-69 3. Inoue,M .,Kishimoto,A.,Takai,Y.,and Nishizuka,Y.(1977) J.Biol.Chtam.252,7610-7616 1012
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4. Hashimoto,E.,Yamamura,H.(l989) J.Biochem.l06,1041-1048 5. Nishizuka,Y.(1986) Science 233,305-312 6. Tapley,P.,Murray,A.,W.(l984) Biochem.Biophys,Res.Commun. 118,835-841 7. Kraft,A.S.,Anderson,W.B.(1983) Nature 301.621-623 8. Melloni,E.,Pontremoli,S.,Michetti,M.,Sacco,O., Sparatore,B., Horecker,B.L.(1986) J.Biol.Chem.261,4101-4105 9. Pontremoli,S.,Michetti,M.,Melloni,E.,Sparatore,B.,Salamino,F., Horecker,B.L.(1990) Proc.Nat1.Acad.Sci.U.S. 87,3705-3707 lO.Taylor,M.V.,Metcalfe,J.C.,Hesketh,T.R.,Smith,G.A., Moore,J.P. (1984) Nature,312,462-465 ll.de Chaffoy de Courcelles,D.,Roevens,P.,van Belle,H.(1984) FEBS Lett. 173,389-393 12.Halenda,S.P.,Feinstein,M.B.(1984) Biochem.Biophys.Res. Commun.24,507-513 13.Shragin,A.S.,Parnova,R.G.,Selichcheva,A.A.,Herman,A.L., Klyaschitsky,B.A.,Shvets,V.I.(1988) The Ukrainian Bi0chem.J. 60,72-77 .,Nishizuka,Y.(198C) Methods 14,Kitano,T.,Go,M.,Kikkawa,I in Enzymology,124,349-353 15.Woodgett,J.R.,Hirnter,T.(1987) J.Biol.Chem. 262,4836-4843 16.Mochly-Rosen,D.,Koshland,D.E.(1987) J.Biol.Chem. 262,2291-2297 17.Lee,M.H.,Bell,R.M.(1986) J.Biol.Chem.261,14867-14870 18.Hashimoto,E.,Yamamura,H.(l989) J,Biochem.,106,1041-1048 19.Cochet,C.,Chambaz,E.M.(1986) Biochem.J.237,25-31 20.Saltiel,A.R.,Fox,J.H.,Sherline,P.,Sahyoun,N., Cuatrecasas,P. (1987) Bi0chem.J. 241,759-763 21.Pike,M.C.,Arndt,C.(l988) J.Immunology. 140,1967-1973 22.Melloni E., Pontremoli S., Michetti M., Sacco O., F. and Horecker B.L. (1985) PrOC. Sparatore B., Salamino Natl.Acad.Sci. USA 82, 6435-6439 V. and Ducastaing A. (1987) 23.Savart M., Belamri M., Pallet FEBS Letters 216, 22-26
1013
176,
No.
May
15,
1991
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
PROTEOLYTIC PHOSPHORYLATES
FRAGMKNT OF PROTEIN KINASE C (KINASE IN VITRO PHOSPHATIDYLINOSITOL-&PHOSPHATE
0. K. Tusupov,
S. E. Severin,*+
1007-1013
M)
and V. I. Shvets
Fine Chemical Technology, M.V. Lomonosov Institute of * Moscow, and Institute of Diabetes, USSR Academy of Medical Science,117036 Moscow, USSR Received
March
15,
1991
Summary: Limited tryptic proteolysis of homogeneous protein kinase C induces the formation of a catalytically active unlike native PK C fragment of 50 kDa (kinase M) which, the ability to phosphorylate PIP. Both ATP and acquires GTP were found to be capable of serving as phosphate donors in this process. Incubation of purified kinase M with a preparation of rat brain membrane fraction enha;;;d the level of phosphorylation of PIP in the presence in the absence of exogenous PIP. A scheme of the interrelationship of phosphoinositide metabolism and the proteolytic processingof protein kinase C is proposed. 01991
Ca2+ ,phospholipid-dependent protein kinase (PK C) is essential for regulatory processes of realizing the cell metabolism resulting from receptor-induced hydrolysis of phosphoinositides (1). One of the products of the phosphoinositide turnover 1,2-diacyl-sn-glycerol (DAG) stimulates protein kinase C, whereas the other, inositol1,4,5trisphosphate (IP3), mobilizes the intracellular Ca 2+. Furthermore, a proteolytically activated form of PK C (protein kinase M (PK M)) that widely occurs in many organs PK C proteolysis and tissues was shown to exist (3). action of calpain after the apparently occurs under the
+To whom correspondence and reprint requests should be addressed at Research Center of Molecular Diagnostics, Simpheropolsky Rlvd., S, 113149 Moscow, USSR. Abbreviations: PK C,protein kinase C;PIP,phosphatidylinositol-I-phosphat; phosphatidylinositol-4,5-bisphosphate; IP3, PIPzr PK M,protein kinase M; TPCK,L-linositol trisphosphate; -chlor-3-tosylamido-4-phenyl-2-butanon. 0006-291X/91
1007
$1.50
Copyight 0 1991 by Accrdernic Press, Inc. All rights of reproduction in any form reserved.
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
enzyme the membrane, activation and translocation to though physiological role of PK M remains unclear the (4,5).Treatment of intact cells with phospholipase C, phorbol esters, chemoattractants, i.e., agents that induce PK C translocation, was shown kinase M accumulation to stimulate (6-9). At the same time, another effect of the above subwas noted, namely the enhancement of stances on the cells lipids (10-12). Our phosphorylation of inositol-containing experimental data on isolation and analysis of phosphatidilinositol-4-phosphate kinase from human brain (unpublished result) and comparison of the enzyme properties with those of the catalytic fragment of PK C (PK M) enabled us to suggest that the latter can perform the function of the PIP kinase. and a hypothesis Evidence in support of the advanced suggestion in the about involvement of proteolytic PK C degradation and regulation of the cell responses are transduction offered below. MATERIALS
AND
Rat
METHODS
brain, line Wistar was used as the source for isolating protein kinase C. [Y-~~PIATP (3000 Ci/mmol) and [Y-~~PIGTP (3000 Ci/mmol) were purchased from Amercham. PIP and PIP2 were obtained as described in (13). All the rest of the reagents were from Sigma and Serva. PK C purification. The enzyme was purified by the ---------------method detailed in (14) or (15). In all cases the preparation was judging by the data homogeneous, of SDS electrophoresis in the system of Laemmli (16), and was a mixture of known PK C isoforms. PK C assay . The activity was assayed as described in (14) -w-w----with slight modifications in the reaction mixture (100 ul) containing 50 mM Tris-HCI buffer pH 7.5, 5 mM MgCI 2 1 mM DTT, 20 ug histone Hl, 0.6 mM CaC12, 50 uM ATP, [T-~~P]ATP (1 uCi), 2 ug PS and 0,2 ug dioleoylglycerol. The lipids were mixed and added into the reaction mixture in the form of sonicated suspension. The basal activity ( PK M activity ) was measured in the presence of 0.7 mM EGTA insteadof dioleoylglycerol, PS and CaC$ !CCyptic of PK C. Trypsin (64 U/mg) was treated ---- proteolysis ---a-----------with loo-fold excess of TPCK for inhibiting chymotrypsin admixtures in the 50 mM Tris-HCI buffer, pH 7.5, containing 0.25 mM EGTA, 0.25 mM EDTA, 0,6 mM CaC12, and purified by gel filtration on PD-10 columm (Pharmacia) in the same buffer, 0.1 mg/ml of the PK C preparation in the buffer of 20 mM Tris-HCI pH 7.5, 0.5 EGTA, 0.5 mM EDTA, 1mM DTT, 0.2 mM NaCI were incubated at 37 Y with TPCK-trypsin at the enzyme-substrate ratio of 1:50 for different time intervals.The reaction was terminated with the lofold excess ( over the trypsin amount ) of soy-bean trypsin inhibitor. The resultant preparation was at once used in experiments on histone and PIP phosphorylation. 1008
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and thin-layer -____________ chromatography---of PIP~~k&z!se-------assay ----------------phosphoinositides. Phosphorylation was carried out in the --- -----------reaction mixture (100 ul) containing 50 mM Tris-HCI pH 7.5, 0.25 mM EDTA, 0.25 mM EGTA, 1 mM DTT, 0.1 M NaCI, 20 mM MgCIZ O,Ol% Triton X-100, 10 mM PIP and 50uM ATP [Y-~~P]ATP (2uCi) .The reaction was initiated by the addition of ATP and, following incubation for 5 min at room temperature (25 4, terminated through introducing into the reaction mixture of 0.3 ml of chloroform, 0.5 ml of the metanol-1N HCI (1:l) solution and 50 ~1 (10 )1g) of cold PIP2 in the chloroform-methanol (2:l) solution. After vigorous stirring, the tubes were centrifuged at 5000 rpm for 5 min for phase separation, whereupon the upper water phase was removed and the lower one was evaporated in a Speedvac concentrator. The evaporated lipids were dissolved in chloroform-methanol (2:1), applied to the plates with silica gel impregnated with 1% potassium oxalate solution, and chromatographed in the n-propanol-4N ammonia (2:l) system. Following autoradiography, the spots corresponding to the position of PIP2 standard were collected from the plate into vials, supplemented with 1 ml of the methanol-1N HCI (1:l) mixture, 9 ml of scintillation cocktail was added and radioactivity was measured in a liquid scintillation spectrometer. RESULTS
AND
DISCUSSION
Fig.1 shows the results of the in vitro experiment with a PK C preparation, which involved the following steps. Highly purified PK C was subjected to limited proteolysis with trypsin, resulting in the formation of a catalytic and a regulatory domain. The process has been described in the literature many times and the products of tryptic proteolysis have been characteAfter the reaction termination with a rized in details (16-18). trypsin inhibitor, optimal conditions were maintained in the system for manifestation of PIP kinase activity (19-21), i.e., Ca2+ was increased. Thereafter ions were bound and Mg2+ concentration the PIP suspension was added and the phosphorylation reaction was was run in painitiated by introducing ATP. The same procedure rallel for native PK C as well. As clear from Fig.lA, native PK C is incapable of phosphorylating PIP, whereas the trypsin-treated enzyme acquires such capacity. Moreover, the maximal level of is achieved as early as within 3 32P i incorporation into PIP 2 min. The following decrease in the label incorporation is well explained by the known fact of trypsin-effected PK C cleavage into smaller fragments that lose the enzymic activity. In a para3s i into histone Hl llel experiment (Fig.lB), incorporation of of native and trypsin-treated PK C was measured. The experimental results indicate that in the result of tryptic proteolysis, Ca2+ ,phospholipid-dependent activity of histone phosphorylation 1009
Vol.
176,
No.
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-
PL 3717
-
354%
-
t
72%
207%
;1 100%
1
2
3
kl 45%
4
5
33
L
2
3
into PIP (A) and histone Fiaure l."- P incorporation H1 (B) 1, protein kinase C forms.A: Purified frown [y- '-P]ATP by different rat brain PK C (5yg) was incubated in conditions for PIP-kinase assay as described in Materials and Methods; control without enzyme (lane l), native enzyme (lane Z),enzyme incubated with 5 respectively). B: PK C trypsin lmin, 3min, 5min (lane 3, 4, (5pg) was incubated in conditions for PK C assay as described in Materials and Methods; native enzyme (lane 1), enzyme incubated with trypsin lmin, 3min (lane 2,3 respectively).
and the basal activity ( kinase M activity ) accordingly increases (Fig.lB). Since PK C is known to be incapable of using GTP as a phosphate donor, while PIP-kinase has been shown to the possess this capacity (19,21), we performed an experiment similar to the above described, except that ATP was substituted for GTP. The experimental results (Fig.2) evidence that the native PK C in the presence of GTP has no potency in phosphorylating PIP, whereas cleavage with trypsin promotes the emergence of this function (Fig.ZA). Meanwhile, neither the native enzyme nor the trypsintreated one can phosphorylate histone, once GTP is used (Fig.2B). The above findings allow a conclusion that the catalytic PK C fragment (kinase M) is able to perform the function of PIPkinase. As the process of phosphoinositide interconversion occurs in the plasmatic membranes of cells, we analysed the effect of exogenous kinase M on PIP phosphorylation in the crude membrane fraction formed following centrifugation of rat brain homogenate at 105,000 g. The data of Fig.3 demonstrate that incubation of decreases
1010
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
182%
UL i
1
1f
-
2
4
1
2
3
Fiqure 2.32 P incorporation into PIP (A) and histone Hl (B) from protein kinase C forms.A: Purified rat [Y-32 PIGTP by different brain PK C (51J.g) was incubated in conditions for PIP-kinase assay as described in Materials and Methods; control without enzyme (lane l), native enzyme (lane 2),enzyme incubated with trypsin lmin, 3min (lane 3,4 respectively). B: PK C (5fig) was incubated in conditions for PK C assay as described in Materials and Methods: native enzyme (lane l), enzyme incubated with trypsin lmin, 3min (lane 2,3 respectively).
the membrane with a kinase M preparation, obtained after cleavage of homogeneous PK C (15) and subsequent purification of the catalytic fragment by DEAE-cellulose chromatography (16), enhances the phosphate incorporation from ATP into PIP2 both in the presence and in the absence of exogenous PIP.
2421
I
PIP
+
+ +
PK n
Piqure 3. Enhancement of 32P incorporation phosphatidylinositol-4-phosphat from crude purified kinase M.
1011
+
from [Y-~~PIATP membrane fraction
into by
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Fioure 4. Proposed scheme interrelationship PK C and phosphoinosiPIP2(step l), turnover: receptor-dependent hydrolys tide 2), protein kinase C and enhancement intracellular Ca ++ (step C and calpain translocation (step 3), proteolysis protein kinase phosphorylation of phosphatidylinositol-4-phosphate by kinase M.
Proceeding from the data of this study as well as known facts that both PK C and calpain in the presence of Ca2+ ions are mainly located on the membrane (22) and, moreover, calpain can be associated with PK C (23), we propose the following hypothetical scheme of interrelationship between PK C and PIP-kinase. Activation of phosphatidylinositol-specific phospholipase C via a corresponding cell receptor leads to a sharp decrease in the PIP2 2t concentration and elevation of Ca , which, in turn, causes PK C translocation to the membrane structures and activation of phosphorylation. A simultaneous activation of calpain will take the latter, while cleaving native PK C, increases the place, kinase M level in the near-membrane region. The enzyme will promote restoration of PIP2 In other words, the proteolytic fragment of PK C acts as a feedback in the case of receptordependent PIP2 degradation (Fig.4). REFERENCES 1. Nishizuka,Y.(1988) Nature 334,661-665 2. Streb,M.D.,Irvine,R.F.,Berridge,M.J.,Schulz,J.(l983) Nature 306, 67-69 3. Inoue,M .,Kishimoto,A.,Takai,Y.,and Nishizuka,Y.(1977) J.Biol.Chtam.252,7610-7616 1012
Vol.
176,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
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