Eur. J. Biochem. 208, 547-557 (1992) 0 FEBS 1992
Review The protein kinase C family Angelo AZZI, Daniel BOSCOBOINIK and Carmel HENSEY Institut fur Biochemie und Molekularbiologie der Universitat Bern, Switzerland (Received April 9/May 19, 1992) - EJB 92 0507
Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-l,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.
The eukaryotic cell is a highly regulated entity, responding to its immediate intracellular environment as well as to external stimuli. Most regulations are mediated, either directly or indirectly, by conformational changes in proteins, and the equilibrium between active and inactive conformational states can be altered by both allosteric and covalent mechanisms. A most common covalent means of regulating protein activity is protein phosphorylation, which is particularly prominent for the role that it serves in signal transduction and is important in many other cellular responses (Edelman et al., 1987). Signals impinging on cells have their effects amplified and distributed by a network of protein phosphorylation and dephosphorylation reactions. Correspondence to A. Azzi, Institut fur Biochemie und Molekularbiologie, Universitat Bern, Buhlstrasse 28, CH-3012 Bern, Switzerland Fax: + 41 31 65 37 37. Abbreviations. EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; ICs0, concentration of a compound giving 50% inhibition; MAP, microtubule-associated protein; MARKS, myristoylated alanine-rich C-kinase substrate; nPKC, novel protein kinases C (&, E - , t-, q-PKC isoenzymes); PDBt, phorbol 12,13dibutyrate; PKC, protein kinase C; PKM, catalytic fragment of protein kinase C ; PtdIns(4))P, phosphatidylinositol 4-monophosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine. Enzymes. Protein kinase (EC 2.7.1.37); calpain (EC 3.4.22.7); phospholipase A2 (EC 3.1.1.4); phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); phosphatidate phosphatase (EC 3.1.3.4); protein-tyrosine kinase (EC 2.7.1.1 12); phosphatidylinositol4-phosphate kinase (EC 2.7.1.68).
The family of enzymes catalyzing the phosphorylation of proteins, the protein kinases, and their complementary protein phosphatases, represent a group of enzymes that modulate the biological activity of proteins in a rapid and reversible manner (Hunter, 1987; Cohen and Cohen, 1989). Two general classes of kinase exist in eukaryotes, those transferring phosphate to serine or threonine residues and those transferring phosphate to tyrosine residues. When the first report on protein kinase C (PKC) appeared in 1977, this cytoplasmic, proteolytically activated protein kinase (Inoue et al., 1977; Takai et al., 1977) had no obvious role in signal transduction. Later, several groups have shown this enzyme to be calcium-activated, phospholipid-dependent (Takai et al., 1979a), and firmly linked to signal transduction. In fact diacylglycerol, an early product of signal-induced inosito1 phospholipid breakdown, greatly increased the affinity of PKC for calcium thereby activating it (Takai et al., 1979b). Phorbol esters can promote tumors and substitute for diacylglycerol in PKC activation (Castagna et al., 1982). Activation of PKC by phorbol esters may be responsible, at least in part, for their tumor-promoting activity (Ashendel, 1985). A review in this field, after those by Nishizuka (1988, 1989), Parker et al. (1989), Berry and Nishizuka (1990), Stabel and Parker (1991) and Housley (1991) appears justified since the central biological interest of the PKC family is far from being exhausted. Moreover, the enormous number of publications on the subject (approximately 40- 50 each week), containing sometimes apparently contradictory information,
548 requires a continuous effort to summarize and simplify the field.
THE PROTEIN KINASE C FAMILY Molecular heterogeneity PKC is a serinelthreonine kinase composed of a single polypeptide chain of 77 - 83 kDa. Enzymatic studies and molecular cloning analyses have revealed that PKC molecules consist of a protein family, which can be classified into two major groups, based on their structure and enzymatic properties, conventional PKCs and novel PKCs (nPKC) (Osada et al., 1990). Several isoenzymes have been defined, which are derived both from multiple genes and alternative splicing of a single RNA transcript. Primary structures of the PKC family were inferred from cDNA sequence determinations and nine members of the PKC family have been identified to date. Initially four cDNA clones that encode a-, PI-, PII- and y-PKC isoenzymes (conventional PKCs) were identified (Parker et al., 1986). The genes for a-, fl- and y-PKC are located on different chromosomes (Parker et al., 1986). Partial genomic analysis suggested that PI-PKC and PII-PKC derive from a single RNA transcript by alternative splicing (Coussens et al., 1986, 1987). Another group of PKC isoenzymes (nPKCs), have also been identified. cDNA clones for a further three isoenzymes, designated 6-, c- and t-PKC, were isolated from a rat brain library (On0 et al., 1987). There is evidence for the alternative splicing of the E-PKC gene since a cDNA clone encoding a truncated version of the PKC enzyme (E’-PKC) lacking 240 N-terminal amino acids present in the E-isoenzyme, has been isolated from rat brain (On0 et al., 1988). Shorter transcripts that differ from E-PKC at the 5’ end have also been reported in rat lung and brain (Schaap et al., 3990a). More recently, two further cDNA clones encoding PKC isoenzymes have been characterized. q-PKC and L-PKC, isolated from a mouse epidermis and a human keratinocyte cDNA library respectively (Osada et al., 1990; Bacher et al., 1991), are related to the 6-, E - and t-isoenzymes of PKC. L-PKC is the human homolog of q-PKC. Differential tissue-specific and functional expression Using a combination of biochemical, immunological and cytochemical procedures with isoenzyme-specific antibodies, the relative activity and individual pattern of expression of multiple PKC isoenzymes in several cells and tissues has been examined (reviewed by Nishizuka, 1988). y-PKC is apparently expressed solely in the brain and spinal cord and is not found in other tissues and cell types (Shearman et al., 1987). The isoenzyme is differentially expressed in various regions of the brain. PI- and PIr-PKC also display differential expression in the brain (Coussens et al., 1987)and are found in many other tissues and a-PKC is widely distributed in many tissues and cell types (Kosaka et al., 1988). In rat brain synaptosomes, PII-isoenzymeis associated with membrane-skeleton elements (Tanaka et al., 1991). The distribution of the 6-, E - and 5-isoenzymes of PKC is less well known but these isoenzymes have been identified in brain and several other tissues including heart, liver, kidney and lung (On0 et al., 1988). The transcript for PKC L is most abundant in lung tissue, less expressed in heart and skin tissue and exhibited very low expression in brain tissue (Bacher et al., 1991). q-PKC
is also highly expressed in lung and skin and only slightly expressed in brain tissue (Osada et al., 1990). The existence of a family of PKC molecules of which more than one isoenzyme is usually expressed in a particular cell type suggests that distinct PKC isoenzymes may activate different cellular pathways and phosphorylate different substrates. This could explain, at least in part, the diversity of responses observed upon PKC activation. The biological role of molecular heterogeneity and differences in expression of PKC isoenzymes is far from being fully understood. Several reports show some specific function associated with given isoenzymes or groups of them. Thyrotropin-releasing hormone selectively down-modulates E-PKC with no effect on aor fl-PKCs (Kiley et al., 1990). Differential regulation of PKC isoenzymes by thyrotropin-releasing hormone may be linked with the selective compartmentalization of E-PKC rendering this pool uniquely susceptible to proteolytic degradation (Kiley et al., 1991). Mischak et al. (1991) have shown that the expression of PKC isoenzymes in hemopoietic cells is cell-type and B cell-differentiation stage-specific. Aihara et al. (1991) have shown that the a-isoenzymes play a key role in HL-60 cell differentiation to macrophages. PKC isoenzymes are also modulated during granulocytic differentiation (Makowske et al., 1988; Hashimoto et al., 1990) and in rat glial cell cultures (Masliah et al., 1991). PKC isoenzymes E- and a-, but little or no q-PKC, are present in murine erythroleukemia cells (Powell et al., 1992). An interesting report of Borner et al. (1990) shows that fibroblast transformation by the ras oncogene increased the expression of M-PKC, and decreased the expression of E-PKC both seen at the protein and mRNA level.
THE PRIMARY STRUCTURE The primary structure of the isoenzymes of PKC shows conserved structural motifs with a high degree of sequence homology. The group of a-, PI-, PI,- and y-isoenzymes have four conserved sections of the polypeptide chain (C, - C,) and five variable ones (V1-V,) (Coussens et al., 1986) (Fig. 1). The nPKC family lacks the C2 section. The aminoterminal half of each polypeptide, containing C1 and C2, as well as V1, V2 and part of V3, is the regulatory domain (intended as the folded polypeptide section) that interacts with Ca2+,phospholipid and diacylglycerol or phorbol ester. The regulatory domain also contains the pseudo-substrate, an amino acid sequence that closely resembles PKC substrate recognition sites. The carboxy-terminal half containing the sections C3, C4 and V4 forms the catalytic domain. The regulatory domain The C1 section The C1 polypeptide section of all PKCs except 5-PKC contains a tandem repeat of a cysteine-rich sequence (Parker et al., 1986) (Fig. 2).
Review The protein kinase C family Angelo AZZI, Daniel BOSCOBOINIK and Carmel HENSEY Institut fur Biochemie und Molekularbiologie der Universitat Bern, Switzerland (Received April 9/May 19, 1992) - EJB 92 0507
Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-l,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.
The eukaryotic cell is a highly regulated entity, responding to its immediate intracellular environment as well as to external stimuli. Most regulations are mediated, either directly or indirectly, by conformational changes in proteins, and the equilibrium between active and inactive conformational states can be altered by both allosteric and covalent mechanisms. A most common covalent means of regulating protein activity is protein phosphorylation, which is particularly prominent for the role that it serves in signal transduction and is important in many other cellular responses (Edelman et al., 1987). Signals impinging on cells have their effects amplified and distributed by a network of protein phosphorylation and dephosphorylation reactions. Correspondence to A. Azzi, Institut fur Biochemie und Molekularbiologie, Universitat Bern, Buhlstrasse 28, CH-3012 Bern, Switzerland Fax: + 41 31 65 37 37. Abbreviations. EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; ICs0, concentration of a compound giving 50% inhibition; MAP, microtubule-associated protein; MARKS, myristoylated alanine-rich C-kinase substrate; nPKC, novel protein kinases C (&, E - , t-, q-PKC isoenzymes); PDBt, phorbol 12,13dibutyrate; PKC, protein kinase C; PKM, catalytic fragment of protein kinase C ; PtdIns(4))P, phosphatidylinositol 4-monophosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine. Enzymes. Protein kinase (EC 2.7.1.37); calpain (EC 3.4.22.7); phospholipase A2 (EC 3.1.1.4); phospholipase C (EC 3.1.4.3); phospholipase D (EC 3.1.4.4); phosphatidate phosphatase (EC 3.1.3.4); protein-tyrosine kinase (EC 2.7.1.1 12); phosphatidylinositol4-phosphate kinase (EC 2.7.1.68).
The family of enzymes catalyzing the phosphorylation of proteins, the protein kinases, and their complementary protein phosphatases, represent a group of enzymes that modulate the biological activity of proteins in a rapid and reversible manner (Hunter, 1987; Cohen and Cohen, 1989). Two general classes of kinase exist in eukaryotes, those transferring phosphate to serine or threonine residues and those transferring phosphate to tyrosine residues. When the first report on protein kinase C (PKC) appeared in 1977, this cytoplasmic, proteolytically activated protein kinase (Inoue et al., 1977; Takai et al., 1977) had no obvious role in signal transduction. Later, several groups have shown this enzyme to be calcium-activated, phospholipid-dependent (Takai et al., 1979a), and firmly linked to signal transduction. In fact diacylglycerol, an early product of signal-induced inosito1 phospholipid breakdown, greatly increased the affinity of PKC for calcium thereby activating it (Takai et al., 1979b). Phorbol esters can promote tumors and substitute for diacylglycerol in PKC activation (Castagna et al., 1982). Activation of PKC by phorbol esters may be responsible, at least in part, for their tumor-promoting activity (Ashendel, 1985). A review in this field, after those by Nishizuka (1988, 1989), Parker et al. (1989), Berry and Nishizuka (1990), Stabel and Parker (1991) and Housley (1991) appears justified since the central biological interest of the PKC family is far from being exhausted. Moreover, the enormous number of publications on the subject (approximately 40- 50 each week), containing sometimes apparently contradictory information,
548 requires a continuous effort to summarize and simplify the field.
THE PROTEIN KINASE C FAMILY Molecular heterogeneity PKC is a serinelthreonine kinase composed of a single polypeptide chain of 77 - 83 kDa. Enzymatic studies and molecular cloning analyses have revealed that PKC molecules consist of a protein family, which can be classified into two major groups, based on their structure and enzymatic properties, conventional PKCs and novel PKCs (nPKC) (Osada et al., 1990). Several isoenzymes have been defined, which are derived both from multiple genes and alternative splicing of a single RNA transcript. Primary structures of the PKC family were inferred from cDNA sequence determinations and nine members of the PKC family have been identified to date. Initially four cDNA clones that encode a-, PI-, PII- and y-PKC isoenzymes (conventional PKCs) were identified (Parker et al., 1986). The genes for a-, fl- and y-PKC are located on different chromosomes (Parker et al., 1986). Partial genomic analysis suggested that PI-PKC and PII-PKC derive from a single RNA transcript by alternative splicing (Coussens et al., 1986, 1987). Another group of PKC isoenzymes (nPKCs), have also been identified. cDNA clones for a further three isoenzymes, designated 6-, c- and t-PKC, were isolated from a rat brain library (On0 et al., 1987). There is evidence for the alternative splicing of the E-PKC gene since a cDNA clone encoding a truncated version of the PKC enzyme (E’-PKC) lacking 240 N-terminal amino acids present in the E-isoenzyme, has been isolated from rat brain (On0 et al., 1988). Shorter transcripts that differ from E-PKC at the 5’ end have also been reported in rat lung and brain (Schaap et al., 3990a). More recently, two further cDNA clones encoding PKC isoenzymes have been characterized. q-PKC and L-PKC, isolated from a mouse epidermis and a human keratinocyte cDNA library respectively (Osada et al., 1990; Bacher et al., 1991), are related to the 6-, E - and t-isoenzymes of PKC. L-PKC is the human homolog of q-PKC. Differential tissue-specific and functional expression Using a combination of biochemical, immunological and cytochemical procedures with isoenzyme-specific antibodies, the relative activity and individual pattern of expression of multiple PKC isoenzymes in several cells and tissues has been examined (reviewed by Nishizuka, 1988). y-PKC is apparently expressed solely in the brain and spinal cord and is not found in other tissues and cell types (Shearman et al., 1987). The isoenzyme is differentially expressed in various regions of the brain. PI- and PIr-PKC also display differential expression in the brain (Coussens et al., 1987)and are found in many other tissues and a-PKC is widely distributed in many tissues and cell types (Kosaka et al., 1988). In rat brain synaptosomes, PII-isoenzymeis associated with membrane-skeleton elements (Tanaka et al., 1991). The distribution of the 6-, E - and 5-isoenzymes of PKC is less well known but these isoenzymes have been identified in brain and several other tissues including heart, liver, kidney and lung (On0 et al., 1988). The transcript for PKC L is most abundant in lung tissue, less expressed in heart and skin tissue and exhibited very low expression in brain tissue (Bacher et al., 1991). q-PKC
is also highly expressed in lung and skin and only slightly expressed in brain tissue (Osada et al., 1990). The existence of a family of PKC molecules of which more than one isoenzyme is usually expressed in a particular cell type suggests that distinct PKC isoenzymes may activate different cellular pathways and phosphorylate different substrates. This could explain, at least in part, the diversity of responses observed upon PKC activation. The biological role of molecular heterogeneity and differences in expression of PKC isoenzymes is far from being fully understood. Several reports show some specific function associated with given isoenzymes or groups of them. Thyrotropin-releasing hormone selectively down-modulates E-PKC with no effect on aor fl-PKCs (Kiley et al., 1990). Differential regulation of PKC isoenzymes by thyrotropin-releasing hormone may be linked with the selective compartmentalization of E-PKC rendering this pool uniquely susceptible to proteolytic degradation (Kiley et al., 1991). Mischak et al. (1991) have shown that the expression of PKC isoenzymes in hemopoietic cells is cell-type and B cell-differentiation stage-specific. Aihara et al. (1991) have shown that the a-isoenzymes play a key role in HL-60 cell differentiation to macrophages. PKC isoenzymes are also modulated during granulocytic differentiation (Makowske et al., 1988; Hashimoto et al., 1990) and in rat glial cell cultures (Masliah et al., 1991). PKC isoenzymes E- and a-, but little or no q-PKC, are present in murine erythroleukemia cells (Powell et al., 1992). An interesting report of Borner et al. (1990) shows that fibroblast transformation by the ras oncogene increased the expression of M-PKC, and decreased the expression of E-PKC both seen at the protein and mRNA level.
THE PRIMARY STRUCTURE The primary structure of the isoenzymes of PKC shows conserved structural motifs with a high degree of sequence homology. The group of a-, PI-, PI,- and y-isoenzymes have four conserved sections of the polypeptide chain (C, - C,) and five variable ones (V1-V,) (Coussens et al., 1986) (Fig. 1). The nPKC family lacks the C2 section. The aminoterminal half of each polypeptide, containing C1 and C2, as well as V1, V2 and part of V3, is the regulatory domain (intended as the folded polypeptide section) that interacts with Ca2+,phospholipid and diacylglycerol or phorbol ester. The regulatory domain also contains the pseudo-substrate, an amino acid sequence that closely resembles PKC substrate recognition sites. The carboxy-terminal half containing the sections C3, C4 and V4 forms the catalytic domain. The regulatory domain The C1 section The C1 polypeptide section of all PKCs except 5-PKC contains a tandem repeat of a cysteine-rich sequence (Parker et al., 1986) (Fig. 2).
