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GTP-binding proteins as possible targets for protein kinase C action
TIBS 14 - September 1989
355
Open Question GTP-Binding proteins as possible targets for protein kinase C action Ronit Sagi-Eisenberg The a-subunits of two guanine nucleotide binding proteins Gi and transducin, as well as the 13-subunit of transducin, serve as substrates for phosphorylation by the Ca2+- and phospholipid-dependent protein kinase C (PKC). Phosphorylation of the a-subunit of transducin is strictly dependent on its conformation and it is only the inactive form that is subjected to phosphorylation by PKC. This review will focus on the proposition that G proteins may serve as cellular targets for modulatory actions of PKC. Guanine nucleotide binding proteins (G proteins) are a highly conserved family of proteins that couple receptors to numerous effector system:~. There is functional evidence that many biological processes may be regulated by G proteins, including the activation and inhibition of adenylate cyclase, activation of a cGMP-phosphodiesterase and phospholipases A2, C and D, modulation of ion channels and exocytosis (reviewed in Refs l-3). The G proteins that have been identified so far share a high degree of structural homology. They are all heterotrimeric, comprising ct-, 13- and 7-subunits. The mechanism by which these', proteins activate their effector systems can be summarized simplistically as follows: the GDP-bound G proteins have an affinity for iigand-bound receptors and on binding, the GDP is exchanged for GTP. This exchange disengages the receptor from the G protein 4 which dissociates into subunits 5, yielding an active GTP-bound a-subunit with a functional life determined by its intrinsic GTPase activity6. The end product of this process is a GDP bound c~-subunit which binds to the ]37-subunits to form a recycled GDP-bound G protein.
Conformational changes duriLagthe G protein cycle An important feature of this G protein cycle is the marked change in the conformation of the c~-subunit that occurs. The structure of the ct-subunit differs depending on whether it is complexed to GDP or to GTP 7. This is R. Sagi-Eisenberg is at the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.
illustrated by the observation that in its GDP-bound form, the ct-subunit has high affinity for the 137-subunits as well as for the receptor. In contrast the GTP-bound form has a reduced affinity for the receptor and ]37-subunits but a high affinity for the effector system. Furthermore, binding of GTP or its non-hydrolysable analogs (e.g. GppNHp) alters the cleavage pattern of the c~-subunit produced by trypsin.
G proteins as programmable messengers The c~-subunits of the regulatory G proteins undergo covalent modifications such as ADP ribosylation by bacterial toxins. These modifications result in changes in their function (reviewed in Refs l-3). The conformation of the G protein apparently affects its ability to serve as a substrate for such modifications. While the holo G protein, in its inactive conformation, serves as a substrate for pertussis toxin, it is the active GTP-bound form of the tt-subunit that is modified by cholera toxin. Furthermore, these covalent modifications appear to stabilize the different conformation states. Pertussis-toxin-catalysed ADP ribosylation prevents subunit dissociation and preserves the G protein in its holo inactive form. In contrast, ADP ribosylation by cholera toxin inhibits GTPase activity thereby stabilizing the G protein in its active conformation. It has been proposed ~ that the G protein c~-subunits may be subjected to additional covalent modifications. According to this attractive hypothesis, these modifications could differently affect G protein functions thus turning the G protein into a 'programmable messenger'.
Protein phosphorylation is the most widely occurring post-translational modification generally used to control biological processes 9. Protein phosphorylation is assumed to alter the structure and function of proteins thereby providing an universal mechanism by which external signals may modulate intracellular events m. This idea has recently gained direct evidence with the demonstration that phosphorylation of the enzyme glycogen phosphorylase results in changes in its structure that are depicted by crystallographic analysis m. Hence, phosphorylation of G proteins could serve as a mechanism to regulate G protein function. Moreover, it is predicted that, like ADP ribosylation, phosphorylation may occur in a conformationdependent manner.
Phosphorylation of G proteins by PKC The ct-subunits of both G~ and the retinal G protein transducin (TD) were shown to serve as in vitro substrates of protein kinase C (PKC) 11"12. In both studies, the holo protein served as a poorer substrate for the kinase than did the purified u-subunits. Phosphoamino acid analysis of TD revealed that phosphorylation occurred exclusively on serine residues 12. To test whether the conformation of the u-subunit of TD (TD,) could affect its susceptibility to serve as substrate for phosphorylation by PKC, purified TD,~ was presented to the kinase either in its GDP or GTP7S-bound forms. These experiments demonstrated that TD~ serves as a substrate for PKC only when presented in its GDP-bound form. In contrast, when liganded with GTPTS, TD~ fails to undergo phosphorylation 12 (Fig. 1). Furthermore, A1F4 and vanadate, agents that have been shown 13't4 to confer an active conformation upon the GDP-bound form of TD,, also inhibit its phosphorylation by PKC 15. Treatment of holo Gi with GTPTS also inhibits its phosphorylation despite the fact that it causes dissociation of the oligomer 11. Thus, taken together, these results suggest that phosphorylation of the c~-subunit does depend on its conformation. While the inactive conformation serves as a high affinity substrate of PKC, the active conformation fails to do so. Similar conformation-dependent
(~) 1989, Elsevier Science Publishers Ltd, (UK)
0376 5067/89/$03.511
356
TIBS 14-September 1989
b
0
c
d i~iij:)i¸:
l
I:1_ I-CD
C 0 C
'qPTD
!i
I r~ t--
I-
Fig. 1. Phosphorylation o f holo transducin (TD) and its subunits by protein kinase C (PK('). Purified holo TD or its isolated subunits were phosphorylated by P K C in the presence o f Ca-'+ (2 raM) and phosphatidylserine (100 ~g ml i). Lanes: a, buffer; b, T D , - G T P y S (3.3 ~g); c, TD (3.3 ~g); d, T D , - G D P (3.3 ~tg). Reproduced from Ref. 12.
phosphorylation of T D , by PKC could also be demonstrated using intact rod outer segment (ROS) membranes, where we could show 15 that purified PKC binds to ROS membranes in a Ca~-+-dependent manner and phosphorylates the endogenous inactive TD,~ as well as several other ROS proteins. Addition of GTPyS resulted in complete inhibition of TD,~ phosphorylation, while not inhibiting phosphorylation of other ROS proteins. This selective conformation -dependent phosphorylation of T D , appears to be
I
PKC ~
137
GDP
Pi
Conclusions Taken together, these findings suggest that G protein functions may be regulated through phosphorylation. By this mechanism external ligands that activate PKC may synergize with or antagonize the action of other hor-
~
H
(z ~
physiologically relevant since it occurs only at a specific point during the G protein cycle (Fig. 2). On the basis of the assumed substrate requirements for PKC phosphorylation, a serine that is located in close proximity to a basic amino acid should serve as the site for phosphorylation within the a subunit. A plausible candidate is Serl2 of TD,~ (Ref. 16), which is conserved in the ct-subunits of both Gj and TD. It is adjacent to several basic amino acids and it is located in the N-terminal end of TD,, that, together with the C-terminal region, plays a role in binding the 137subunits 17q9. In the holo protein, TD3r binds to this region and may thus disturb its phosphorylation II Furthermore, the purified TDf~ also serves as a high affinity in vitro substrate of PKC L~. Therefore, a possible functional consequence of phosphorylation of both subunits could be the inhibition of association of the ~- with the [3y-subunits (Fig. 2). Thus, it is predicted that the [3-subunit may also be phosphorylated at a site located in a region that is involved in binding to the c~-subunit. Recent crosslinking experiments Is, indicate that a 5 kDa C-terminal fragment of TD,, directly interacts with a 15 kDa N-terminal fragment of TD~. A possible candidate for phosphorylation by PKC within this region of TD~ would be Ser 74.
Acknowledgements
GDP O{~r. . . . . . . . . . . . .
GTP
I GTP
mones whose receptors arc coupled to G proteins (reviewed in Refs 20, 21). Furthermore, G proteins also serve as in vitro substrates for phosphorylation by protein tyrosine kinases such as the insulin and IGF-I receptor kinases ~2"2e. It is thus possible that phosphorylation could differently affect G protein functions depending on whether phosphorylation takes place on tyrosine or serine residues. Phosphorylation of G proteins could thereby serve as a point of transregulation by different signal transduction mechanisms. These results also demonstrate for the first time that a conformational change of proteins, induced by allosteric effectors, is critical for their susceptibility to serve as substrates of PKC. An analogous situation, where phosphorylation ofa substrate depends on its structure is the agonist-specific phosphorylation of G protein coupled receptors by the 13-adrenergic receptor kinase (13ARK) 23. This kinase phosphorylates specifically the agonist-occupied forms of the 13- and c~2-adrenergic receptors 23"24 resulting in uncoupling of the receptor from the G protein leading to homologous, agonist-specific desensitization. As the antagonist-bound receptors fail to undergo phosphorylation, it is reasonable to postulate that only the active conformation of the receptor that is capable of interacting and activating the respective G protein, is subjected to regulation by [3ARK. Thus, conformation-dependent phosphorylation may serve as a safety mechanism to ascertain its occurrence only when its functional consequences arc required. It will therefore be of interest to determine whether other proteins that play regulatory roles in signal transduction are subjected to phosphorylation by PKC in a conformation-dependent manner.
Fig. 2. Model for phosphorylation o f the ct- and [~-subunits by protein kinase ('.
I am grateful to my collaborators, Drs Israel Pecht, Allen Spiegel and Yehiel Zick for their major contributions. 1 thank Ms Esther Gross for excellent secretarial assistance. RSE is the incumbent of the Charles H. Revson Career Development Award.
References 1 Strycr, L. and Bourne, H. R. (1986) Annu. Rev. Cell Biol. 2,391M 19 2 Spiegel, A. M. (1987) Mol. Cell. Endocrinol. 4% 1-16 3 Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649
TIBS 14- September 1989 4 Brandt, D. R. and Ross, E. M. (1986) J. Biol. Chem. 261, 1656-1664 5 Ransnas, L. A. and lnsel, P. A. ( [988)J. Biol. (;hem. 263, 17239 17242 6 Cassel, D. and Selinger, Z. (1976) Biochem. Biophys. Acta 452, 538 551 7 Fung, B. K-K. andNash, C. R. (i 983) J. Biol. ~7~em. 258, 10503-105 I0 8 Rodbell, M. (1985) Trends Bioellem. Sci. I(1, 461-464 9 Krebs, E. G. (1986) In The Enzy,nes, 3rd Edn Vol. 17(Boyer, P. D. and Krebs. E. G., eds), pp. 3-2(/, Academic Press 10 Sprang, S. R., Acharya, K. R., Goldsmith, E. J., Stuart, D. I., Varvill, K., Flenerick, R. J., Madsen, N. B. and Jolnson, L. N. (1988) Nature 336, 215 221
357 11 Katada, T., Gilman, A. G., Watanabe, Y., Bauer, S. and Jakobs, K. H. (1985) Eur. J. Biochem. 151,431-437 12 Zick, Y., Sagi-Eisenberg, R., Pines, M., Gierschick, P. and Spiegel, A. M. (1986) Proc. Natl Acad. Sci. USA 83, 9294-9297 13 Begay,J., Deterrc, P., Pfieter, C. andChabre, M. (1985)FEBSLett. 191,181 185 14 Kanaho, Y., Chang, P. P., Moss, J. and Vaughan, M. (19881 .I. Biol. Chem. 263, 17584-17589 15 Sagi-Eisenberg, R.,Traub,L.,Spiegel,A. M. and Zick, Y. Cellular Signalling Vol. 1 No. 5 (in press) 16 Lochrie, M. A. and Simon, M. I. (1988) Biochemistry 27, 4957-4965 17 Navon, S. E. and Fung, B. K-K. (1987) J. Biol. (Jwm. 262, 15746-15751
18 Hingorani, V. N., Tobias, D. T., Henderson, J. T. and Ho, Y-K. (1988) J. Biol. Chem. 263, 6916-6926 19 Neer, E. J., Pulsifer, L. and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9(10(I 2(1 Kikkawa, U. and Nishizuka, Y. (1986) Annu. Rev. Cell. Biol. 2,149 178 21 Berridge, M. (1987)Annu. Rev. Biochem. 56, 15%193 22 Zick, Y., Spiegel, A. M. and Sagi-Eisenberg, R. (1987)J. Biol. Chem. 262, 1(1259-10264 23 Benovic, J. L., Strasserm, R. H., Cason, M. G. and Lefkowitz, R. J. (1986) Proc. Natl Acad. Sci. USA 83, 2797-28(11 24 Bcnovic, J. L., Regan, J. W., Matsui, H., Mayor, F., Jr, Cotecchia, S., Leeb-Lundberg, F. L. M., Caron, M. G. and Lefkowitz, R. J. (1987)J. Biol. Chem. 262, 17251 17253
Textbook Error Regulation of vertebrate striated muscle contraction Michael R. Payne and Suzanne E. Rudnick Most current textbooks of ceh biology and histology use the steric blocking model to describe the protein mechanism by which vertebrate striated muscle contraction is regulated. Evidence accumuiated in the past decade, however, reveals the regulation of muscle contraction to be far more complex than this model predicts. The vertebrate striated muscle sarcomere is composed of thin and thick filaments which slide past one another to interdigitate and produce the sarcomere contraction. The thin filament, anchored at the z-line, is tormed by actin (A), tropomyosin ( T M ) a n d three troponins (TN) (Fig. 1). F-actin, a helical two-stranded filament of G-actin monomers, is the backbone of the thin filament ~. Along each of the two grooves created by the F-acti n helix lies a TM filament formed by a head-to-tail polymerization of TM dimers 2. Each TM dimer spans seven actin monomers and the TM filament extends the length of the thin filament. Troponin binds periodically along the TM filament and is a complex of three polypeptides3: TN-C (calcium binding), TN-I (inhibitory) and TN-T (TM binding). The T N - T M proteins form the calciumbinding regulatory complex of vertebrate striated muscle. M. R. Payne is at the Department of Cell Biology and Anatomy, Basic Sciences Btdlding, New York Medical College, Valhalla. N Y 10595, USA. S. E. Rudnick is at the Department c f Chernistry, Manhattan College, Riverdale, N Y 10471, USA.
The thick filament consists of myosin along with several other proteins present in minor quantities. Each myosin consists of two major polypeptides which form both a two-chain c~-helical rod region and two separate elongated globular regions called myosin heads 4. The rod region assembles into a bipolar thick filament. The myosin head contains the ATPase active site, which provides the energy to drive the contraction, as well as the binding site for actin. Myosin can be cleaved by proteolytic enzymes such as papain, which isolates individual myosin heads (myosin S l), and chymotrypsin, which produces a fragment containing both myosin heads and a portion of the rod region (heavy meromyosin, HMM) 4'5. The head region of myosin also has up to three types of lower molecular weight polypeptidcs (myosin light chains) associated with it. The myosin heads bridge the physical distance between the thick and thin filaments and interact cyclically with the actin of the thin filament. When bound to actin, myosin undergoes a rotational or conformational change that results in movement of the thin
filament relative to the thick illament ~m. The precise region in myosin where force generation occurs is not currently known and multiple sites of flexibility may, in fact, be required 1°. The myosin head then detaches and re-attaches to a different site on the actin filament in order to repeat this process. Only a fraction of the available myosin heads actually interact with actin during a contraction. A relative sliding occurs which interdigitates the thick and thin filaments (the sliding filament model), resulting in the shortening of the muscle sarcomere 7'~. During contraction, the rate of ATP hydrolysis increases several hundredfold due to the activation of the myosin ATPase by actin. The effect of calcium Regulation of vertebrate striated muscle contraction occurs with changes in the intracellular free calcium concentration. Calcium is released from the sarcoplasmic rcticulum which surrounds each sarcomere. As the calcium concentration is increased from a p p r o x i m a t e l y 10 - 7 t o 10-5M, TM and the TNs function as a calcium-sensitive regulatory switch. As its concentration rises, calcium binds to TN-C and induces changes within the TN complex which overcome the inhibitory effect of TN-I. Then, through TN-T, a signal is sent to TM which turns on the contractile event 9,1°. With the fall in calcium concentration, as calcium is again sequestered by the sarcoplasmic reticulum, the inhibitory effects of TN-I presumably take over and changes occur in the protein interactions which result in relaxation ')1(I.
© 1989,ElsevierSciencePublisherslad, (UK) 037~ 511(~7/89/$035()
355
Open Question GTP-Binding proteins as possible targets for protein kinase C action Ronit Sagi-Eisenberg The a-subunits of two guanine nucleotide binding proteins Gi and transducin, as well as the 13-subunit of transducin, serve as substrates for phosphorylation by the Ca2+- and phospholipid-dependent protein kinase C (PKC). Phosphorylation of the a-subunit of transducin is strictly dependent on its conformation and it is only the inactive form that is subjected to phosphorylation by PKC. This review will focus on the proposition that G proteins may serve as cellular targets for modulatory actions of PKC. Guanine nucleotide binding proteins (G proteins) are a highly conserved family of proteins that couple receptors to numerous effector system:~. There is functional evidence that many biological processes may be regulated by G proteins, including the activation and inhibition of adenylate cyclase, activation of a cGMP-phosphodiesterase and phospholipases A2, C and D, modulation of ion channels and exocytosis (reviewed in Refs l-3). The G proteins that have been identified so far share a high degree of structural homology. They are all heterotrimeric, comprising ct-, 13- and 7-subunits. The mechanism by which these', proteins activate their effector systems can be summarized simplistically as follows: the GDP-bound G proteins have an affinity for iigand-bound receptors and on binding, the GDP is exchanged for GTP. This exchange disengages the receptor from the G protein 4 which dissociates into subunits 5, yielding an active GTP-bound a-subunit with a functional life determined by its intrinsic GTPase activity6. The end product of this process is a GDP bound c~-subunit which binds to the ]37-subunits to form a recycled GDP-bound G protein.
Conformational changes duriLagthe G protein cycle An important feature of this G protein cycle is the marked change in the conformation of the c~-subunit that occurs. The structure of the ct-subunit differs depending on whether it is complexed to GDP or to GTP 7. This is R. Sagi-Eisenberg is at the Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel.
illustrated by the observation that in its GDP-bound form, the ct-subunit has high affinity for the 137-subunits as well as for the receptor. In contrast the GTP-bound form has a reduced affinity for the receptor and ]37-subunits but a high affinity for the effector system. Furthermore, binding of GTP or its non-hydrolysable analogs (e.g. GppNHp) alters the cleavage pattern of the c~-subunit produced by trypsin.
G proteins as programmable messengers The c~-subunits of the regulatory G proteins undergo covalent modifications such as ADP ribosylation by bacterial toxins. These modifications result in changes in their function (reviewed in Refs l-3). The conformation of the G protein apparently affects its ability to serve as a substrate for such modifications. While the holo G protein, in its inactive conformation, serves as a substrate for pertussis toxin, it is the active GTP-bound form of the tt-subunit that is modified by cholera toxin. Furthermore, these covalent modifications appear to stabilize the different conformation states. Pertussis-toxin-catalysed ADP ribosylation prevents subunit dissociation and preserves the G protein in its holo inactive form. In contrast, ADP ribosylation by cholera toxin inhibits GTPase activity thereby stabilizing the G protein in its active conformation. It has been proposed ~ that the G protein c~-subunits may be subjected to additional covalent modifications. According to this attractive hypothesis, these modifications could differently affect G protein functions thus turning the G protein into a 'programmable messenger'.
Protein phosphorylation is the most widely occurring post-translational modification generally used to control biological processes 9. Protein phosphorylation is assumed to alter the structure and function of proteins thereby providing an universal mechanism by which external signals may modulate intracellular events m. This idea has recently gained direct evidence with the demonstration that phosphorylation of the enzyme glycogen phosphorylase results in changes in its structure that are depicted by crystallographic analysis m. Hence, phosphorylation of G proteins could serve as a mechanism to regulate G protein function. Moreover, it is predicted that, like ADP ribosylation, phosphorylation may occur in a conformationdependent manner.
Phosphorylation of G proteins by PKC The ct-subunits of both G~ and the retinal G protein transducin (TD) were shown to serve as in vitro substrates of protein kinase C (PKC) 11"12. In both studies, the holo protein served as a poorer substrate for the kinase than did the purified u-subunits. Phosphoamino acid analysis of TD revealed that phosphorylation occurred exclusively on serine residues 12. To test whether the conformation of the u-subunit of TD (TD,) could affect its susceptibility to serve as substrate for phosphorylation by PKC, purified TD,~ was presented to the kinase either in its GDP or GTP7S-bound forms. These experiments demonstrated that TD~ serves as a substrate for PKC only when presented in its GDP-bound form. In contrast, when liganded with GTPTS, TD~ fails to undergo phosphorylation 12 (Fig. 1). Furthermore, A1F4 and vanadate, agents that have been shown 13't4 to confer an active conformation upon the GDP-bound form of TD,, also inhibit its phosphorylation by PKC 15. Treatment of holo Gi with GTPTS also inhibits its phosphorylation despite the fact that it causes dissociation of the oligomer 11. Thus, taken together, these results suggest that phosphorylation of the c~-subunit does depend on its conformation. While the inactive conformation serves as a high affinity substrate of PKC, the active conformation fails to do so. Similar conformation-dependent
(~) 1989, Elsevier Science Publishers Ltd, (UK)
0376 5067/89/$03.511
356
TIBS 14-September 1989
b
0
c
d i~iij:)i¸:
l
I:1_ I-CD
C 0 C
'qPTD
!i
I r~ t--
I-
Fig. 1. Phosphorylation o f holo transducin (TD) and its subunits by protein kinase C (PK('). Purified holo TD or its isolated subunits were phosphorylated by P K C in the presence o f Ca-'+ (2 raM) and phosphatidylserine (100 ~g ml i). Lanes: a, buffer; b, T D , - G T P y S (3.3 ~g); c, TD (3.3 ~g); d, T D , - G D P (3.3 ~tg). Reproduced from Ref. 12.
phosphorylation of T D , by PKC could also be demonstrated using intact rod outer segment (ROS) membranes, where we could show 15 that purified PKC binds to ROS membranes in a Ca~-+-dependent manner and phosphorylates the endogenous inactive TD,~ as well as several other ROS proteins. Addition of GTPyS resulted in complete inhibition of TD,~ phosphorylation, while not inhibiting phosphorylation of other ROS proteins. This selective conformation -dependent phosphorylation of T D , appears to be
I
PKC ~
137
GDP
Pi
Conclusions Taken together, these findings suggest that G protein functions may be regulated through phosphorylation. By this mechanism external ligands that activate PKC may synergize with or antagonize the action of other hor-
~
H
(z ~
physiologically relevant since it occurs only at a specific point during the G protein cycle (Fig. 2). On the basis of the assumed substrate requirements for PKC phosphorylation, a serine that is located in close proximity to a basic amino acid should serve as the site for phosphorylation within the a subunit. A plausible candidate is Serl2 of TD,~ (Ref. 16), which is conserved in the ct-subunits of both Gj and TD. It is adjacent to several basic amino acids and it is located in the N-terminal end of TD,, that, together with the C-terminal region, plays a role in binding the 137subunits 17q9. In the holo protein, TD3r binds to this region and may thus disturb its phosphorylation II Furthermore, the purified TDf~ also serves as a high affinity in vitro substrate of PKC L~. Therefore, a possible functional consequence of phosphorylation of both subunits could be the inhibition of association of the ~- with the [3y-subunits (Fig. 2). Thus, it is predicted that the [3-subunit may also be phosphorylated at a site located in a region that is involved in binding to the c~-subunit. Recent crosslinking experiments Is, indicate that a 5 kDa C-terminal fragment of TD,, directly interacts with a 15 kDa N-terminal fragment of TD~. A possible candidate for phosphorylation by PKC within this region of TD~ would be Ser 74.
Acknowledgements
GDP O{~r. . . . . . . . . . . . .
GTP
I GTP
mones whose receptors arc coupled to G proteins (reviewed in Refs 20, 21). Furthermore, G proteins also serve as in vitro substrates for phosphorylation by protein tyrosine kinases such as the insulin and IGF-I receptor kinases ~2"2e. It is thus possible that phosphorylation could differently affect G protein functions depending on whether phosphorylation takes place on tyrosine or serine residues. Phosphorylation of G proteins could thereby serve as a point of transregulation by different signal transduction mechanisms. These results also demonstrate for the first time that a conformational change of proteins, induced by allosteric effectors, is critical for their susceptibility to serve as substrates of PKC. An analogous situation, where phosphorylation ofa substrate depends on its structure is the agonist-specific phosphorylation of G protein coupled receptors by the 13-adrenergic receptor kinase (13ARK) 23. This kinase phosphorylates specifically the agonist-occupied forms of the 13- and c~2-adrenergic receptors 23"24 resulting in uncoupling of the receptor from the G protein leading to homologous, agonist-specific desensitization. As the antagonist-bound receptors fail to undergo phosphorylation, it is reasonable to postulate that only the active conformation of the receptor that is capable of interacting and activating the respective G protein, is subjected to regulation by [3ARK. Thus, conformation-dependent phosphorylation may serve as a safety mechanism to ascertain its occurrence only when its functional consequences arc required. It will therefore be of interest to determine whether other proteins that play regulatory roles in signal transduction are subjected to phosphorylation by PKC in a conformation-dependent manner.
Fig. 2. Model for phosphorylation o f the ct- and [~-subunits by protein kinase ('.
I am grateful to my collaborators, Drs Israel Pecht, Allen Spiegel and Yehiel Zick for their major contributions. 1 thank Ms Esther Gross for excellent secretarial assistance. RSE is the incumbent of the Charles H. Revson Career Development Award.
References 1 Strycr, L. and Bourne, H. R. (1986) Annu. Rev. Cell Biol. 2,391M 19 2 Spiegel, A. M. (1987) Mol. Cell. Endocrinol. 4% 1-16 3 Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649
TIBS 14- September 1989 4 Brandt, D. R. and Ross, E. M. (1986) J. Biol. Chem. 261, 1656-1664 5 Ransnas, L. A. and lnsel, P. A. ( [988)J. Biol. (;hem. 263, 17239 17242 6 Cassel, D. and Selinger, Z. (1976) Biochem. Biophys. Acta 452, 538 551 7 Fung, B. K-K. andNash, C. R. (i 983) J. Biol. ~7~em. 258, 10503-105 I0 8 Rodbell, M. (1985) Trends Bioellem. Sci. I(1, 461-464 9 Krebs, E. G. (1986) In The Enzy,nes, 3rd Edn Vol. 17(Boyer, P. D. and Krebs. E. G., eds), pp. 3-2(/, Academic Press 10 Sprang, S. R., Acharya, K. R., Goldsmith, E. J., Stuart, D. I., Varvill, K., Flenerick, R. J., Madsen, N. B. and Jolnson, L. N. (1988) Nature 336, 215 221
357 11 Katada, T., Gilman, A. G., Watanabe, Y., Bauer, S. and Jakobs, K. H. (1985) Eur. J. Biochem. 151,431-437 12 Zick, Y., Sagi-Eisenberg, R., Pines, M., Gierschick, P. and Spiegel, A. M. (1986) Proc. Natl Acad. Sci. USA 83, 9294-9297 13 Begay,J., Deterrc, P., Pfieter, C. andChabre, M. (1985)FEBSLett. 191,181 185 14 Kanaho, Y., Chang, P. P., Moss, J. and Vaughan, M. (19881 .I. Biol. Chem. 263, 17584-17589 15 Sagi-Eisenberg, R.,Traub,L.,Spiegel,A. M. and Zick, Y. Cellular Signalling Vol. 1 No. 5 (in press) 16 Lochrie, M. A. and Simon, M. I. (1988) Biochemistry 27, 4957-4965 17 Navon, S. E. and Fung, B. K-K. (1987) J. Biol. (Jwm. 262, 15746-15751
18 Hingorani, V. N., Tobias, D. T., Henderson, J. T. and Ho, Y-K. (1988) J. Biol. Chem. 263, 6916-6926 19 Neer, E. J., Pulsifer, L. and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9(10(I 2(1 Kikkawa, U. and Nishizuka, Y. (1986) Annu. Rev. Cell. Biol. 2,149 178 21 Berridge, M. (1987)Annu. Rev. Biochem. 56, 15%193 22 Zick, Y., Spiegel, A. M. and Sagi-Eisenberg, R. (1987)J. Biol. Chem. 262, 1(1259-10264 23 Benovic, J. L., Strasserm, R. H., Cason, M. G. and Lefkowitz, R. J. (1986) Proc. Natl Acad. Sci. USA 83, 2797-28(11 24 Bcnovic, J. L., Regan, J. W., Matsui, H., Mayor, F., Jr, Cotecchia, S., Leeb-Lundberg, F. L. M., Caron, M. G. and Lefkowitz, R. J. (1987)J. Biol. Chem. 262, 17251 17253
Textbook Error Regulation of vertebrate striated muscle contraction Michael R. Payne and Suzanne E. Rudnick Most current textbooks of ceh biology and histology use the steric blocking model to describe the protein mechanism by which vertebrate striated muscle contraction is regulated. Evidence accumuiated in the past decade, however, reveals the regulation of muscle contraction to be far more complex than this model predicts. The vertebrate striated muscle sarcomere is composed of thin and thick filaments which slide past one another to interdigitate and produce the sarcomere contraction. The thin filament, anchored at the z-line, is tormed by actin (A), tropomyosin ( T M ) a n d three troponins (TN) (Fig. 1). F-actin, a helical two-stranded filament of G-actin monomers, is the backbone of the thin filament ~. Along each of the two grooves created by the F-acti n helix lies a TM filament formed by a head-to-tail polymerization of TM dimers 2. Each TM dimer spans seven actin monomers and the TM filament extends the length of the thin filament. Troponin binds periodically along the TM filament and is a complex of three polypeptides3: TN-C (calcium binding), TN-I (inhibitory) and TN-T (TM binding). The T N - T M proteins form the calciumbinding regulatory complex of vertebrate striated muscle. M. R. Payne is at the Department of Cell Biology and Anatomy, Basic Sciences Btdlding, New York Medical College, Valhalla. N Y 10595, USA. S. E. Rudnick is at the Department c f Chernistry, Manhattan College, Riverdale, N Y 10471, USA.
The thick filament consists of myosin along with several other proteins present in minor quantities. Each myosin consists of two major polypeptides which form both a two-chain c~-helical rod region and two separate elongated globular regions called myosin heads 4. The rod region assembles into a bipolar thick filament. The myosin head contains the ATPase active site, which provides the energy to drive the contraction, as well as the binding site for actin. Myosin can be cleaved by proteolytic enzymes such as papain, which isolates individual myosin heads (myosin S l), and chymotrypsin, which produces a fragment containing both myosin heads and a portion of the rod region (heavy meromyosin, HMM) 4'5. The head region of myosin also has up to three types of lower molecular weight polypeptidcs (myosin light chains) associated with it. The myosin heads bridge the physical distance between the thick and thin filaments and interact cyclically with the actin of the thin filament. When bound to actin, myosin undergoes a rotational or conformational change that results in movement of the thin
filament relative to the thick illament ~m. The precise region in myosin where force generation occurs is not currently known and multiple sites of flexibility may, in fact, be required 1°. The myosin head then detaches and re-attaches to a different site on the actin filament in order to repeat this process. Only a fraction of the available myosin heads actually interact with actin during a contraction. A relative sliding occurs which interdigitates the thick and thin filaments (the sliding filament model), resulting in the shortening of the muscle sarcomere 7'~. During contraction, the rate of ATP hydrolysis increases several hundredfold due to the activation of the myosin ATPase by actin. The effect of calcium Regulation of vertebrate striated muscle contraction occurs with changes in the intracellular free calcium concentration. Calcium is released from the sarcoplasmic rcticulum which surrounds each sarcomere. As the calcium concentration is increased from a p p r o x i m a t e l y 10 - 7 t o 10-5M, TM and the TNs function as a calcium-sensitive regulatory switch. As its concentration rises, calcium binds to TN-C and induces changes within the TN complex which overcome the inhibitory effect of TN-I. Then, through TN-T, a signal is sent to TM which turns on the contractile event 9,1°. With the fall in calcium concentration, as calcium is again sequestered by the sarcoplasmic reticulum, the inhibitory effects of TN-I presumably take over and changes occur in the protein interactions which result in relaxation ')1(I.
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