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Phosphorylation of plasma proteins with emphasis on complement component C3
Molecular Immunology 36 (1999) 233±239
Mini Review
Phosphorylation of plasma proteins with emphasis on complement component C3 Kristina Nilsson Ekdahl a, b,*, Bo Nilsson a a
The Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden b The Department of Natural Sciences, University of Kalmar, Kalmar, Sweden
Abstract Phosphorylation of complement component C3 by dierent protein kinases in vitro has been demonstrated to alter the functional properties of the protein. Extracellular phosphorylation mediated by activated platelets is a newly described mechanism by which the function of plasma proteins can be regulated. Upon activation of platelets a casein kinase is released concomitant with large amounts of ATP and Ca2+. These components are sucient to phosphorylate proteins e.g., C3 extracellularly. In vivo, in patients with SLE, the phosphate content in plasma proteins, including C3 has been demonstrated to increase during exacerbation. The changes were linked to platelet activation by a covariation with the levels of bthromboglobulin. The purpose of this review is to summarise the ®ndings in this ®eld. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Complement; In¯ammation; Phosphorylation; Platelets; Protein kinases
1. Protein phosphorylation and speci®city of protein kinases Phosphorylation is a rapid way to alter the properties of a protein, e.g., its enzymatic or co-factor activity, its anity for ligands, or its susceptibility to proteolytic degradation. Protein kinases are enzymes, which catalyze the transfer of the g-phosphoryl group of ATP to an amino acid side chain in the presence of divalent cations. Unlike many other regulatory mechanisms, phosphorylation is a reversible process since the phosphate group(s) may be removed by phosphoprotein phosphatases, thereby restoring the original properties of the protein. One example is ®brinogen which has been phosphorylated by protein kinase C and regains its original gelation properties after the
* Corresponding author.
phosphate has been removed by alkaline phosphatase (Forsberg and Martin, 1990). Most of the known protein kinases belong to a large superfamily of homologous proteins, the two main groups of which are protein kinases that phosphorylate serine or threonine and those which are speci®c for tyrosine (Hanks and Hunter, 1995). Protein kinases are subdivided according to their substrate speci®city and dependence on second messengers. Here, only serine/ threonine protein kinases will be discussed. In mammalian cells, cAMP-dependent protein kinase (PKA) exists in three almost indistinguishable isoforms (Taylor et al., 1990). PKA has a broad speci®city and requires basic amino acid residues on the Nterminal side of the phosphorylated amino acid (Kennely and Krebs, 1991). PKA is activated when cAMP binds to the regulatory subunits of the inactive holoenzyme, thereby releasing the active catalytic subunits (Taylor, 1989). A vast number of isoforms of protein kinase C
0161-5890/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 1 - 5 8 9 0 ( 9 9 ) 0 0 0 3 7 - 1
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(PKC), belonging to at least three dierent groups, with dierent sensitivity to activators have been described in mammalian cells. Initially, PKC was described as dependent on Ca2+ ions, phospholipids, e.g., phosphatidylserine, and diacyl-glycerol (Takai et al., 1979) but later forms of PKC which are independent of Ca2+ have been described (Blobe et al., 1996). Like PKA, PKC requires basic amino acid residues in the vicinity of the phosphorylated amino acid, but its speci®city is less well de®ned (Pearson and Kemp, 1991). The model substrates for PKA and PKC in vitro are histones and protamine. Casein kinases (CKs) are the collective name for a group of three ubiquitous enzymes: protein kinase CK1 (Tuazon and Traugh, 1991), protein kinase CK2 (Pinna and Meggio, 1997), and Golgi CK (Lasa et al., 1997). CK phosphorylate proteins which, like the model substrates casein and phosvitin, contain clusters of acidic amino acids (Kemp, 1990). CK1 and CK2 dier concerning speci®city, structure and response to eectors. CK1 is a monomeric enzyme, which has no known physiological inhibitors. In contrast, CK2 occurs as an oligomer consisting of two catalytic and two non-catalytic subunits (Pinna, 1997) and is sensitive to inhibition by heparin (Hathaway et al., 1980). CK1 is extremely well conserved between dierent species, e.g., with 94% homology between rat and human CK1g2 (Kitabayashi et al., 1997). There is even a considerable degree of homology between mammalian and yeast CK1 (Robinson et al., 1992).
2. Phosphorylation in vitro as a regulator of plasma protein function A number of plasma proteins have been shown to contain covalently bound phosphate in vivo. These include coagulation and complement proteins, e.g., ®brinogen (BlombaÈck et al., 1962), coagulation factor V (Rand et al., 1994), vitronectin (Mehringer et al., 1991), and complement component C3 (Martin, 1989; Nilsson Ekdahl and Nilsson, 1995). We have estimated that plasma proteins in healthy blood donors contain approximately 0.09 mol phosphate per mol average protein (Nilsson Ekdahl et al., 1997). Phosphorylation in vitro of proteins of the complement and coagulation cascades has been demonstrated with dierent protein kinases. Human ®brinogen has been shown to be a substrate for PKA, PKC, CK1, and CK2 (Humble et al., 1985). Phosphorylation of ®brinogen with PKC leads to changes in the thickness of the ®brin bundles generated by thrombin (Forsberg, 1989; Martin et al., 1991). Coagulation factor Va becomes more susceptible to
Fig. 1. Phosphorylation of complement component C3 in vitro. C3 has been demonstrated to be phosphorylated in the C3a-domain by cAMP-dependent protein kinase (PKA; 1) and by Protein kinase C (PKC; 2). An ecto-protein kinase on Leishmania major phosphorylates 71Ser in the C3a domain of human C3 (3). Phosphorylation in the C3a-domain stabilises C3 against cleavage into C3a and C3b. Platelet CK (4) phosphorylates the a-chain of the C3d-domain of C3 close to the thiol-ester. This phosphorylation increases the binding of the protein to the target surface and inhibits the cleavage of the C3ba'-chain by factor I with factor H as co-factor. Membranebound CK2 phosphorylates the a-chain of C3 in the 40 kDa-fragment of the C3c domain (5) and makes C3 more susceptible to leukocyte elastase, which cleaves C3 into an iC3b-like fragment. Additional phosphorylation sites for PKC (2) and CK2 (5) are located in the b-chain.
cleavage by activated protein C after phosphorylation by CK2 from activated human platelets (Kalafatis et al., 1993), and phosphorylation of vitronectin with PKC attenuates its cleavage by plasmin (Gechtman and Shaltiel, 1997). C9 was shown to be phosphorylated by ecto-protein kinases on human leukemia cells, e.g., U937 and K562 (Fishelson et al., 1989). These cells were demonstrated to shed microparticles with functionally intact ectoprotein kinase from the cell membrane (Paas and Fishelson, 1995). The main protein kinase activity on these microparticles was later identi®ed as a CK2 (Nilsson Ekdahl and Nilsson, 1997). C3 has been shown to be phosphorylated in vitro by at least ®ve protein kinases, in dierent domains of the molecules and with dierent functional impacts (Fig. 1). Both PKA and PKC phosphorylate a serine residue in the basic C3a domain of C3 (Forsberg et al., 1990), and an ecto-protein kinase on Leishmania major was shown to phosphorylate 71Ser in C3a (Hermoso et al., 1991). PKA and PKC only phosphorylate native C3 but not iC3 or C3a (Forsberg et al., 1990). The eect of phosphorylation of C3 by these protein kinases was to protect the molecule against cleavage at the 77 Arg±78Ser bond, either by trypsin (all three) or by both the classical and the alternative pathway convertase (PKA and PKC). It is not known whether phosphorylation in any way aects the activity of C3a. In addition to the phosphorylation in C3a, PKC also
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Fig. 2. Phosphorylation of complement component C3 in vivo at several sites by dierent protein kinases. (a) C3 can be phosphorylated extracellularly in whole blood by a platelet casein kinase (PL CK) which is released when platelets are activated, e.g., by immune complexes (IC), C5b-9 complexes, ADP or thrombin. Upon activation, the ATP and cations in the dense granulae (black) and the proteins in the a-granulae (white) are released. (b) In monocytes, phosphorylation of C3 can mediated by casein kinase type 2 (CK2). (c) After synthesis in HepG2 cells C3 is phosphorylated by a protein kinase which is inhibited by Staurosporine, most likely protein kinase C (PKC).
phosphorylated at least one threonine residue in the bchain (Forsberg et al., 1990). We have studied the synthesis and phosphorylation of C3 in intact HepG2 cells (Fig. 2). By using a pulsechase technique we could detect phosphorylated intact pro-C3 intracellularly, while the mature C3 which was released consisted of phosphorylated a-chain and unlabelled b-chain (K. Nilsson Ekdahl and B. Nilsson, unpublished observation). Phosphorylation of C3 was almost totally abolished in HepG2 cells, which had been exposed to Staurosporine, which is a potent inhibitor of PKC (Barendsen et al., 1990). This observation suggests that PKC may have a physiological role in the regulation of C3 activity. C3 has also been shown to be a substrate for dierent forms of the acidic amino acid dependent casein kinases. Puri®ed C3 was phosphorylated in vitro by using CK2 on microparticles, which were shed from U937 cells (Fig. 1). CK2 phosphorylated both polypeptide chains of intact C3 to approximately the same degree, and increased its susceptibility to cleavage by elastase (Nilsson Ekdahl and Nilsson, 1997). In intact U937 cells, a fraction of the C3 was phosphorylated intracellularily before release (Fig. 2). During release, the phosphorylated C3 was quantitatively cleaved into
iC3b-like fragments, while nonphosphorylated C3 was released in its intact form. Stimulation of CD11b/ CD18 on the cells increased the fraction of phosphorylated C3 by up to 7-fold (Nilsson Ekdahl and Nilsson, 1997). In vitro, C3 has been shown to be phosphorylated in both chains by recombinant rat CK1 (K. Nilsson Ekdahl and B. Nilsson, unpublished observation). The functional impact(s) of phosphorylation by CK1 is under investigation. Finally, we have demonstrated that C3 was phosphorylated in vitro, in the C3d-region, by a CK which was released from human platelets by physiological activation (Figs. 1 and 2; Nilsson Ekdahl and Nilsson, 1995). This CK1-like protein kinase which was found to be distinct from CK2 is discussed in greater detail below.
3. The platelet as a mediator of extracellular phosphorylation in vivo Mammalian blood cells, in particular platelets, con-
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tain PKA and PKC (Kawahara et al., 1980). KorcGrodzicki et al. (1988a, b) and McGuire et al. (1988) described a PKA in plasma, which phosphorylated vitronectin. Platelets have been shown to contain other types of protein kinases which are involved in a number of intracellular processes (Ferrell and Martin, 1989; Wang et al., 1993; Wiedmer and Sims, 1991; Naik et al., 1991). In contrast, extracellular phosphorylation mediated by activated platelets, which release protein kinases, is a newly described mechanism by which the function of plasma proteins can be regulated. Kalafatis et al. (1998, 1993) have identi®ed a membrane bound CK2 released by activated platelets which phosphorylated coagulation factors Va and VIIIa. We have described a soluble CK which was released from platelets under similar conditions and which phosphorylated human C3. This was not a CK2 because it was not inhibited by heparin (Nilsson Ekdahl and Nilsson, 1995). The similarities between platelet CK and CK1 were investigated regarding eect of speci®c protein kinase inhibitors, immunological reactivity and dependence on divalent cations. Platelet CK was partially inhibited by compounds speci®c for CK1, but with a 10-fold higher Ki than reported for CK1, suggesting similarity but not identity between the two enzymes. This interpretation was supported by a weak crossreactivity between antibodies raised against CK1 and platelet CK. Both protein kinases phosphorylated C3 and casein, but while Mn2+ potentiated phosphorylation by platelet CK, it inhibited phosphorylation by CK1. Taken together, these results suggest that the platelet protein kinase is a CK, which is distinct from CK1 (Nilsson Ekdahl and Nilsson, 1999). Platelet CK is released from activated platelets concomitant with large amounts of ATP and Ca2+. The dense granulae contain Ca2+ and ATP in the 0.5 M range (Holmsen, 1992). When platelets in blood are activated the ATP level increases from 2 to 20 pmol/mL (Nilsson Ekdahl and Nilsson, 1995). In the near vicinity of the activated platelets the concentration of these substances can be assumed to be substantially higher. It was con®rmed that the platelet content of ATP and cations are sucient to phosphorylate proteins, since activation of puri®ed platelets resulted in the phosphorylation of exogenously added C3. Furthermore, C3 was phosphorylated in whole blood upon activation of platelets by ADP or thrombin without any further additives (Nilsson Ekdahl et al., 1997). To test the hypothesis that plasma proteins were phosphorylated extracellularly we investigated patients with SLE. These patients are known to experience platelet activation during exacerbation (Robey, 1994; Sims and Wiedmer, 1991; Zoli et al.,
1995). It was demonstrated that the phosphate content of plasma proteins as a whole and of C3 and ®brinogen in particular increased in parallel to platelet activation assessed as release of b-thromboglobulin (Nilsson Ekdahl et al., 1997). Taken together, these observations combined with the fact that platelets release ATP, calcium and a CK, strongly suggest that phosphorylation of plasma proteins occurs in vivo due to platelet activation.
4. Phosphorylation of C3 by platelet CK Phosphorylation of C3 with platelet CK in the C3d domain inhibited factor I dependent inactivation of C3b into iC3b which represents 2 sequential cleavages at 1281Arg±1282Ser and 1298Arg±1299Ser in the a '-chain (Nilsson Ekdahl and Nilsson, 1995). This observation led to speculations as to whether phosphorylation of C3 might increase the amount of C3b which binds to a target surface, e.g., an immune complex. This was indeed seen when serum from a patient de®cient in C3 was reconstituted with phosphorylated or unphosphorylated C3 and then incubated on an IgG-coated surface. Phosphorylation increased the amount of generated C3 fragments which bound to IgG more than fourfold (Nilsson Ekdahl and Nilsson, 1999). However, the substantial increase in binding of C3 fragments was accompanied by a more modest increase in C3a generation, suggesting additional mechanisms for the increased binding. Exposure of phosphorylated C3 to the classical pathway convertase on EAC14oxy2 cells resulted in approximately two-fold higher binding of C3 fragments as compared to unphosphorylated C3. It is unlikely that the increased binding was due to increased cleavage, since the alternative convertase and trypsin which cleave C3 at the same peptide bond, are unaected by this phosphorylation. Instead we proposed that the dierence in binding was caused by an increase in the number of available acceptor sites for phosphorylated C3. This hypothesis was supported by the observation of signi®cantly (approximately 1.6 times) higher binding of fragments generated from phosphorylated C3 by cleavage by either the C3bBb convertase or by trypsin to surface-bound IgG (Nilsson Ekdahl and Nilsson, 1999). The putative phosphorylation site for platelet CK in C3 is located within a tryptic fragment, between 979 Lys and 1014Lys, which also comprises the thiol ester. In this fragment 1009Thr in the sequence DETEQWE (from de Bruijn and Fey, 1985) is surrounded by several acidic amino acid residues which
K. Nilsson Ekdahl, B. Nilsson / Molecular Immunology 36 (1999) 233±239
makes it a potential phosphorylation site for CKs (Kemp, 1990). One possible explanation for the dierence in binding eciency is that phosphorylation of C3 in some way alters the binding properties of the thiol ester. To test this possibility, phosphorylated or unphosphorylated C3 was cleaved by trypsin in the presence of either (2-3H)glycerol or (2-3H)glycine (Dodds and Law, 1990). It was seen that phosphorylation of C3 increased the glycerol binding capacity (approximately 1.6 times) while it decreased the glycine binding, resulting in an increased ratio of glycerol/glycine binding from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. This could account for the increased binding of C3 fragments to IgG since it has been demonstrated that the binding of C3b to IgG mainly occurs by ester bonds which are formed with hydroxyl group containing amino acid residues in the heavy chain of IgG (Sahu and Pangburn, 1994, 1995; Shohet et al., 1993). In the recently published crystal structure of human C3d (Nagar et al., 1998) it is con®rmed that 38Thr of C3d (which corresponds to 1009 Thr of intact C3) is exposed on the exterior of the molecule in close proximity to a cluster of acidic amino acid residues. The mechanism by which phosphorylation of 38Thr alters the binding properties of C3 has not been established; one possibility is that the negatively charged phosphate group alters the distance between the thiol ester and 133His thereby further favoring the formation of ester bond instead of amides. In addition to C3, we have reported that ®brinogen, vitronectin, and human serum albumin become phosphorylated, both in vitro by platelet CK and in vivo in SLE patients (Nilsson Ekdahl et al., 1997). 5. Implications for extracellular phosphorylation Extracellular phosphorylation is a newly described regulator of plasma proteins. Two CKs have been described to be involved in the process but other protein kinases may also contribute. C3 and other complement components are substrates for platelet CK. Phosphorylation has profound eects on C3 function including eects on proteolytic processes and binding to a target. However, it is likely that platelet mediated phosphorylation is at least as potent regulator of the functions of coagulation proteins. Kalafatis et al. (1993) have shown that factor Va and factor VIIIa are phosphorylated by platelet CK2 which enhances the cleavage of factor Va by activated protein C. Furthermore, we have shown that factor XI becomes more susceptible to cleavage by factor XIIa and by thrombin after phosphorylation by platelet CK (Nilsson Ekdahl et al., 1999). These ®ndings suggest that platelets are
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able to amplify the coagulation cascade via the intrinsic pathway. Further studies are needed to map the consequences of platelet phosphorylation. Acknowledgements We thank Mrs Foozieh Ghazi for her excellent technical assistance and Professor Rolf Larsson for critical reading of the manuscript. This study was supported by grants from the GoÈran Gustafsson Research Foundation, King Gustaf V's Research Foundation, The Swedish Rheumatism Association, Professor Nanna Svartz' Research Foundations, the Swedish Board for Industrial and Technical Development, and by grants nos. 5647 and 11578 from the Swedish Society of Medicine.
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Mini Review
Phosphorylation of plasma proteins with emphasis on complement component C3 Kristina Nilsson Ekdahl a, b,*, Bo Nilsson a a
The Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden b The Department of Natural Sciences, University of Kalmar, Kalmar, Sweden
Abstract Phosphorylation of complement component C3 by dierent protein kinases in vitro has been demonstrated to alter the functional properties of the protein. Extracellular phosphorylation mediated by activated platelets is a newly described mechanism by which the function of plasma proteins can be regulated. Upon activation of platelets a casein kinase is released concomitant with large amounts of ATP and Ca2+. These components are sucient to phosphorylate proteins e.g., C3 extracellularly. In vivo, in patients with SLE, the phosphate content in plasma proteins, including C3 has been demonstrated to increase during exacerbation. The changes were linked to platelet activation by a covariation with the levels of bthromboglobulin. The purpose of this review is to summarise the ®ndings in this ®eld. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Complement; In¯ammation; Phosphorylation; Platelets; Protein kinases
1. Protein phosphorylation and speci®city of protein kinases Phosphorylation is a rapid way to alter the properties of a protein, e.g., its enzymatic or co-factor activity, its anity for ligands, or its susceptibility to proteolytic degradation. Protein kinases are enzymes, which catalyze the transfer of the g-phosphoryl group of ATP to an amino acid side chain in the presence of divalent cations. Unlike many other regulatory mechanisms, phosphorylation is a reversible process since the phosphate group(s) may be removed by phosphoprotein phosphatases, thereby restoring the original properties of the protein. One example is ®brinogen which has been phosphorylated by protein kinase C and regains its original gelation properties after the
* Corresponding author.
phosphate has been removed by alkaline phosphatase (Forsberg and Martin, 1990). Most of the known protein kinases belong to a large superfamily of homologous proteins, the two main groups of which are protein kinases that phosphorylate serine or threonine and those which are speci®c for tyrosine (Hanks and Hunter, 1995). Protein kinases are subdivided according to their substrate speci®city and dependence on second messengers. Here, only serine/ threonine protein kinases will be discussed. In mammalian cells, cAMP-dependent protein kinase (PKA) exists in three almost indistinguishable isoforms (Taylor et al., 1990). PKA has a broad speci®city and requires basic amino acid residues on the Nterminal side of the phosphorylated amino acid (Kennely and Krebs, 1991). PKA is activated when cAMP binds to the regulatory subunits of the inactive holoenzyme, thereby releasing the active catalytic subunits (Taylor, 1989). A vast number of isoforms of protein kinase C
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(PKC), belonging to at least three dierent groups, with dierent sensitivity to activators have been described in mammalian cells. Initially, PKC was described as dependent on Ca2+ ions, phospholipids, e.g., phosphatidylserine, and diacyl-glycerol (Takai et al., 1979) but later forms of PKC which are independent of Ca2+ have been described (Blobe et al., 1996). Like PKA, PKC requires basic amino acid residues in the vicinity of the phosphorylated amino acid, but its speci®city is less well de®ned (Pearson and Kemp, 1991). The model substrates for PKA and PKC in vitro are histones and protamine. Casein kinases (CKs) are the collective name for a group of three ubiquitous enzymes: protein kinase CK1 (Tuazon and Traugh, 1991), protein kinase CK2 (Pinna and Meggio, 1997), and Golgi CK (Lasa et al., 1997). CK phosphorylate proteins which, like the model substrates casein and phosvitin, contain clusters of acidic amino acids (Kemp, 1990). CK1 and CK2 dier concerning speci®city, structure and response to eectors. CK1 is a monomeric enzyme, which has no known physiological inhibitors. In contrast, CK2 occurs as an oligomer consisting of two catalytic and two non-catalytic subunits (Pinna, 1997) and is sensitive to inhibition by heparin (Hathaway et al., 1980). CK1 is extremely well conserved between dierent species, e.g., with 94% homology between rat and human CK1g2 (Kitabayashi et al., 1997). There is even a considerable degree of homology between mammalian and yeast CK1 (Robinson et al., 1992).
2. Phosphorylation in vitro as a regulator of plasma protein function A number of plasma proteins have been shown to contain covalently bound phosphate in vivo. These include coagulation and complement proteins, e.g., ®brinogen (BlombaÈck et al., 1962), coagulation factor V (Rand et al., 1994), vitronectin (Mehringer et al., 1991), and complement component C3 (Martin, 1989; Nilsson Ekdahl and Nilsson, 1995). We have estimated that plasma proteins in healthy blood donors contain approximately 0.09 mol phosphate per mol average protein (Nilsson Ekdahl et al., 1997). Phosphorylation in vitro of proteins of the complement and coagulation cascades has been demonstrated with dierent protein kinases. Human ®brinogen has been shown to be a substrate for PKA, PKC, CK1, and CK2 (Humble et al., 1985). Phosphorylation of ®brinogen with PKC leads to changes in the thickness of the ®brin bundles generated by thrombin (Forsberg, 1989; Martin et al., 1991). Coagulation factor Va becomes more susceptible to
Fig. 1. Phosphorylation of complement component C3 in vitro. C3 has been demonstrated to be phosphorylated in the C3a-domain by cAMP-dependent protein kinase (PKA; 1) and by Protein kinase C (PKC; 2). An ecto-protein kinase on Leishmania major phosphorylates 71Ser in the C3a domain of human C3 (3). Phosphorylation in the C3a-domain stabilises C3 against cleavage into C3a and C3b. Platelet CK (4) phosphorylates the a-chain of the C3d-domain of C3 close to the thiol-ester. This phosphorylation increases the binding of the protein to the target surface and inhibits the cleavage of the C3ba'-chain by factor I with factor H as co-factor. Membranebound CK2 phosphorylates the a-chain of C3 in the 40 kDa-fragment of the C3c domain (5) and makes C3 more susceptible to leukocyte elastase, which cleaves C3 into an iC3b-like fragment. Additional phosphorylation sites for PKC (2) and CK2 (5) are located in the b-chain.
cleavage by activated protein C after phosphorylation by CK2 from activated human platelets (Kalafatis et al., 1993), and phosphorylation of vitronectin with PKC attenuates its cleavage by plasmin (Gechtman and Shaltiel, 1997). C9 was shown to be phosphorylated by ecto-protein kinases on human leukemia cells, e.g., U937 and K562 (Fishelson et al., 1989). These cells were demonstrated to shed microparticles with functionally intact ectoprotein kinase from the cell membrane (Paas and Fishelson, 1995). The main protein kinase activity on these microparticles was later identi®ed as a CK2 (Nilsson Ekdahl and Nilsson, 1997). C3 has been shown to be phosphorylated in vitro by at least ®ve protein kinases, in dierent domains of the molecules and with dierent functional impacts (Fig. 1). Both PKA and PKC phosphorylate a serine residue in the basic C3a domain of C3 (Forsberg et al., 1990), and an ecto-protein kinase on Leishmania major was shown to phosphorylate 71Ser in C3a (Hermoso et al., 1991). PKA and PKC only phosphorylate native C3 but not iC3 or C3a (Forsberg et al., 1990). The eect of phosphorylation of C3 by these protein kinases was to protect the molecule against cleavage at the 77 Arg±78Ser bond, either by trypsin (all three) or by both the classical and the alternative pathway convertase (PKA and PKC). It is not known whether phosphorylation in any way aects the activity of C3a. In addition to the phosphorylation in C3a, PKC also
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Fig. 2. Phosphorylation of complement component C3 in vivo at several sites by dierent protein kinases. (a) C3 can be phosphorylated extracellularly in whole blood by a platelet casein kinase (PL CK) which is released when platelets are activated, e.g., by immune complexes (IC), C5b-9 complexes, ADP or thrombin. Upon activation, the ATP and cations in the dense granulae (black) and the proteins in the a-granulae (white) are released. (b) In monocytes, phosphorylation of C3 can mediated by casein kinase type 2 (CK2). (c) After synthesis in HepG2 cells C3 is phosphorylated by a protein kinase which is inhibited by Staurosporine, most likely protein kinase C (PKC).
phosphorylated at least one threonine residue in the bchain (Forsberg et al., 1990). We have studied the synthesis and phosphorylation of C3 in intact HepG2 cells (Fig. 2). By using a pulsechase technique we could detect phosphorylated intact pro-C3 intracellularly, while the mature C3 which was released consisted of phosphorylated a-chain and unlabelled b-chain (K. Nilsson Ekdahl and B. Nilsson, unpublished observation). Phosphorylation of C3 was almost totally abolished in HepG2 cells, which had been exposed to Staurosporine, which is a potent inhibitor of PKC (Barendsen et al., 1990). This observation suggests that PKC may have a physiological role in the regulation of C3 activity. C3 has also been shown to be a substrate for dierent forms of the acidic amino acid dependent casein kinases. Puri®ed C3 was phosphorylated in vitro by using CK2 on microparticles, which were shed from U937 cells (Fig. 1). CK2 phosphorylated both polypeptide chains of intact C3 to approximately the same degree, and increased its susceptibility to cleavage by elastase (Nilsson Ekdahl and Nilsson, 1997). In intact U937 cells, a fraction of the C3 was phosphorylated intracellularily before release (Fig. 2). During release, the phosphorylated C3 was quantitatively cleaved into
iC3b-like fragments, while nonphosphorylated C3 was released in its intact form. Stimulation of CD11b/ CD18 on the cells increased the fraction of phosphorylated C3 by up to 7-fold (Nilsson Ekdahl and Nilsson, 1997). In vitro, C3 has been shown to be phosphorylated in both chains by recombinant rat CK1 (K. Nilsson Ekdahl and B. Nilsson, unpublished observation). The functional impact(s) of phosphorylation by CK1 is under investigation. Finally, we have demonstrated that C3 was phosphorylated in vitro, in the C3d-region, by a CK which was released from human platelets by physiological activation (Figs. 1 and 2; Nilsson Ekdahl and Nilsson, 1995). This CK1-like protein kinase which was found to be distinct from CK2 is discussed in greater detail below.
3. The platelet as a mediator of extracellular phosphorylation in vivo Mammalian blood cells, in particular platelets, con-
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tain PKA and PKC (Kawahara et al., 1980). KorcGrodzicki et al. (1988a, b) and McGuire et al. (1988) described a PKA in plasma, which phosphorylated vitronectin. Platelets have been shown to contain other types of protein kinases which are involved in a number of intracellular processes (Ferrell and Martin, 1989; Wang et al., 1993; Wiedmer and Sims, 1991; Naik et al., 1991). In contrast, extracellular phosphorylation mediated by activated platelets, which release protein kinases, is a newly described mechanism by which the function of plasma proteins can be regulated. Kalafatis et al. (1998, 1993) have identi®ed a membrane bound CK2 released by activated platelets which phosphorylated coagulation factors Va and VIIIa. We have described a soluble CK which was released from platelets under similar conditions and which phosphorylated human C3. This was not a CK2 because it was not inhibited by heparin (Nilsson Ekdahl and Nilsson, 1995). The similarities between platelet CK and CK1 were investigated regarding eect of speci®c protein kinase inhibitors, immunological reactivity and dependence on divalent cations. Platelet CK was partially inhibited by compounds speci®c for CK1, but with a 10-fold higher Ki than reported for CK1, suggesting similarity but not identity between the two enzymes. This interpretation was supported by a weak crossreactivity between antibodies raised against CK1 and platelet CK. Both protein kinases phosphorylated C3 and casein, but while Mn2+ potentiated phosphorylation by platelet CK, it inhibited phosphorylation by CK1. Taken together, these results suggest that the platelet protein kinase is a CK, which is distinct from CK1 (Nilsson Ekdahl and Nilsson, 1999). Platelet CK is released from activated platelets concomitant with large amounts of ATP and Ca2+. The dense granulae contain Ca2+ and ATP in the 0.5 M range (Holmsen, 1992). When platelets in blood are activated the ATP level increases from 2 to 20 pmol/mL (Nilsson Ekdahl and Nilsson, 1995). In the near vicinity of the activated platelets the concentration of these substances can be assumed to be substantially higher. It was con®rmed that the platelet content of ATP and cations are sucient to phosphorylate proteins, since activation of puri®ed platelets resulted in the phosphorylation of exogenously added C3. Furthermore, C3 was phosphorylated in whole blood upon activation of platelets by ADP or thrombin without any further additives (Nilsson Ekdahl et al., 1997). To test the hypothesis that plasma proteins were phosphorylated extracellularly we investigated patients with SLE. These patients are known to experience platelet activation during exacerbation (Robey, 1994; Sims and Wiedmer, 1991; Zoli et al.,
1995). It was demonstrated that the phosphate content of plasma proteins as a whole and of C3 and ®brinogen in particular increased in parallel to platelet activation assessed as release of b-thromboglobulin (Nilsson Ekdahl et al., 1997). Taken together, these observations combined with the fact that platelets release ATP, calcium and a CK, strongly suggest that phosphorylation of plasma proteins occurs in vivo due to platelet activation.
4. Phosphorylation of C3 by platelet CK Phosphorylation of C3 with platelet CK in the C3d domain inhibited factor I dependent inactivation of C3b into iC3b which represents 2 sequential cleavages at 1281Arg±1282Ser and 1298Arg±1299Ser in the a '-chain (Nilsson Ekdahl and Nilsson, 1995). This observation led to speculations as to whether phosphorylation of C3 might increase the amount of C3b which binds to a target surface, e.g., an immune complex. This was indeed seen when serum from a patient de®cient in C3 was reconstituted with phosphorylated or unphosphorylated C3 and then incubated on an IgG-coated surface. Phosphorylation increased the amount of generated C3 fragments which bound to IgG more than fourfold (Nilsson Ekdahl and Nilsson, 1999). However, the substantial increase in binding of C3 fragments was accompanied by a more modest increase in C3a generation, suggesting additional mechanisms for the increased binding. Exposure of phosphorylated C3 to the classical pathway convertase on EAC14oxy2 cells resulted in approximately two-fold higher binding of C3 fragments as compared to unphosphorylated C3. It is unlikely that the increased binding was due to increased cleavage, since the alternative convertase and trypsin which cleave C3 at the same peptide bond, are unaected by this phosphorylation. Instead we proposed that the dierence in binding was caused by an increase in the number of available acceptor sites for phosphorylated C3. This hypothesis was supported by the observation of signi®cantly (approximately 1.6 times) higher binding of fragments generated from phosphorylated C3 by cleavage by either the C3bBb convertase or by trypsin to surface-bound IgG (Nilsson Ekdahl and Nilsson, 1999). The putative phosphorylation site for platelet CK in C3 is located within a tryptic fragment, between 979 Lys and 1014Lys, which also comprises the thiol ester. In this fragment 1009Thr in the sequence DETEQWE (from de Bruijn and Fey, 1985) is surrounded by several acidic amino acid residues which
K. Nilsson Ekdahl, B. Nilsson / Molecular Immunology 36 (1999) 233±239
makes it a potential phosphorylation site for CKs (Kemp, 1990). One possible explanation for the dierence in binding eciency is that phosphorylation of C3 in some way alters the binding properties of the thiol ester. To test this possibility, phosphorylated or unphosphorylated C3 was cleaved by trypsin in the presence of either (2-3H)glycerol or (2-3H)glycine (Dodds and Law, 1990). It was seen that phosphorylation of C3 increased the glycerol binding capacity (approximately 1.6 times) while it decreased the glycine binding, resulting in an increased ratio of glycerol/glycine binding from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. This could account for the increased binding of C3 fragments to IgG since it has been demonstrated that the binding of C3b to IgG mainly occurs by ester bonds which are formed with hydroxyl group containing amino acid residues in the heavy chain of IgG (Sahu and Pangburn, 1994, 1995; Shohet et al., 1993). In the recently published crystal structure of human C3d (Nagar et al., 1998) it is con®rmed that 38Thr of C3d (which corresponds to 1009 Thr of intact C3) is exposed on the exterior of the molecule in close proximity to a cluster of acidic amino acid residues. The mechanism by which phosphorylation of 38Thr alters the binding properties of C3 has not been established; one possibility is that the negatively charged phosphate group alters the distance between the thiol ester and 133His thereby further favoring the formation of ester bond instead of amides. In addition to C3, we have reported that ®brinogen, vitronectin, and human serum albumin become phosphorylated, both in vitro by platelet CK and in vivo in SLE patients (Nilsson Ekdahl et al., 1997). 5. Implications for extracellular phosphorylation Extracellular phosphorylation is a newly described regulator of plasma proteins. Two CKs have been described to be involved in the process but other protein kinases may also contribute. C3 and other complement components are substrates for platelet CK. Phosphorylation has profound eects on C3 function including eects on proteolytic processes and binding to a target. However, it is likely that platelet mediated phosphorylation is at least as potent regulator of the functions of coagulation proteins. Kalafatis et al. (1993) have shown that factor Va and factor VIIIa are phosphorylated by platelet CK2 which enhances the cleavage of factor Va by activated protein C. Furthermore, we have shown that factor XI becomes more susceptible to cleavage by factor XIIa and by thrombin after phosphorylation by platelet CK (Nilsson Ekdahl et al., 1999). These ®ndings suggest that platelets are
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able to amplify the coagulation cascade via the intrinsic pathway. Further studies are needed to map the consequences of platelet phosphorylation. Acknowledgements We thank Mrs Foozieh Ghazi for her excellent technical assistance and Professor Rolf Larsson for critical reading of the manuscript. This study was supported by grants from the GoÈran Gustafsson Research Foundation, King Gustaf V's Research Foundation, The Swedish Rheumatism Association, Professor Nanna Svartz' Research Foundations, the Swedish Board for Industrial and Technical Development, and by grants nos. 5647 and 11578 from the Swedish Society of Medicine.
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