Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis (2000)

Protein kinase C (PKC) is a multifunctional, cyclic nucleotide-independent protein kinase that phosphorylates serine and threonine residues in many target proteins. This enzyme was identified in bovine cerebellum by Nishizuka and co-workers (Takai et al., 1977; Inoue et al., 1977) as a protein kinase that phosphorylated histone and protamine. Since its discovery, much interest has been shown in PKC and its role in signal transduction. Development (Otte et al., 1991), memory (Alkon, 1989), differentiation (Cutler et al., 1993), proliferation (Murray et al., 1993) and carcinogenesis (Ashendel, 1985) all are processes for which PKC has been implicated. Once thought to be a single protein, PKC is now known to comprise a large family of enzymes that differ in structure, cofactor requirements and function. Indeed, the PKC family is the largest serine/threonine-specific kinase family known (Parker, 1992) to which many cellular responses have been credited (Nishizuka, 1995). This enzyme multiplicity, together with variation in cellular and tissue distribution, and abundance might explain why so many signal transduction functions have been attributed to this kinase. Here we briefly describe the organization and regulation of PKC and review the current understanding of their role in the regulation of airways smooth muscle (ASM) tone and mitogenesis, which have been investigated in some detail. PKC represents a structurally homologous group of proteins of which at present 10 isoforms have been identified unequivocally. The PKC family can be broadly divided into three groups that differ in their cofactor requirements. These are known as conventional (c)PKC isoforms (α, βI, βII and γ), that require Ca2+ and diacylglycerol (DAG) to become activated; novel (n)PKC isoforms (δ, ε, ζ, θ and μ) that require only DAG; and the atypical (a)PKC isoforms, namely ζ, ι and λ (the mouse homologue of human PKCι), that require neither Ca2+ nor DAG. All PKC family members possess a phosphatidylserine (PS) binding domain for membrane interaction (though the specific lipid activators of aPKC' are not yet known). The general structure of a PKC molecule consists of a catalytic and a regulatory domain found at the C- and N-terminus respectively. Both domain structures are composed of a number of conserved regions (C1–C4) interspersed with regions of lower homology, so-called variable domains, V0–V5 (Figure 1). Diagrammatic representation of the domain structure of PKC isoenzymes. The PKC family can be divided into three sub-families consisting of cPKC', namely α, β1, β2 and γ, the nPKC' namely δ, ε, ζ, θ and μ that do not require Ca2+ (as they do not possess the C2 domain) and the aPKC', namely ζ, ι (human) and λ (mouse) that require neither Ca2+ nor DAG for activation. The length and relative sizes of the isoforms are given to demonstrate the variability within this kinase family. V regions represent poorly conserved (variable) sequences between the isoforms and separate the highly conserved functionally-specific domains. The V3 region acts as a link between the regulatory and catalytic domains and also provides a site for proteolytic cleavage by calpains and trypsin, which results in constitutive and activator-dependent kinase activity (Mochly-Rosen et al., 1990). V0 is an N-terminal domain not present in the cPKC'. The C domains are conserved sequences throughout the PKC family. C1 features the DAG and PS binding sites. At the N-terminus of the C1 domain is located a pseudosubstrate site that regulates PKC activity. These 'so-called' autoinhibitory domains contain a consensus phosphorylation sequence but no phosphorylatable residue, thus controlling kinase activity by blocking access of the substrate through occupation of the catalytic site (Soderling, 1990). C2 is the Ca2+-binding domain present only in cPKC'. The C-terminus of all PKCs features the highly conserved catalytic domain (the C3 and C4 regions), separated by a short variable (V4) insert present only in PKCγ (Parker, 1992). Various sub-domains within the C3-C4 regions have also been identified (Hanks et al., 1988). The C3 region, which is essential for enzyme activity, represents the conserved ATP-binding site sequence found in all isoforms in which an invariant lysine residue (lys-380) is essential for kinase activity (Freisewinkel et al., 1991). The C4 domain represents the catalytic core of PKC'. For further details on the structure of PKC, interested readers should refer to Azzi et al. (1992), Hug & Sarre (1993), Stabel & Parker (1991), Dekker & Parker (1994) and Mellor & Parker (1998). The most recently discovered members of the 'PKC-superfamily' are the protein kinase C-related kinases (PRKs), which display sequence homology with PKC' and, like aPKC', are insensitive to Ca2+, DAG and phorbol diesters. These enzymes were discovered independently by Palmer et al. (1994) and Kitagawa et al. (1995) (who named it PKN) utilizing PCR and low-stringency screening, and are regulated by small GTP-binding proteins such as Rho. Indeed, PRK' belong to the Rho-activated kinase family that include rhotekin and rhophilin (see Mellor & Parker, 1998). Another novel serine/threonine protein kinase that binds phorbol diesters was identified in 1994 by Valverde and colleagues but was not classified into a particular kinase family. This enzyme shows greatest homology (41%) with myosin light chain kinase (MLCK) (Valverde et al., 1994) and was named protein kinase D (PKD), although it may represent the mouse equivalent of human PKCμ (which at 916 amino acids is six residues shorter than PKD) or of an undiscovered human homologue of human PKCμ (Johannes et al., 1994). However, the 54 amino acid difference between PKD and PKCμ exceeds the number seen for other isoforms when inter-species comparisons are made and considerable controversy, thus, surrounds the issue of whether PKD/PKCμ are true PKC family members (see Ron & Kazanietz, 1999). Finally, protein kinase B (also known as RAC-PK (related to A- & C-kinase) was identified as a serine/threonine protein kinase having a catalytic domain resembling PKC and PKA (hence the nomenclature) (Coffer & Woodgett, 1991; Jones et al., 1991). This kinase has been implicated in a number of signalling pathways, most recently in those controlling ASM proliferation (Walker et al., 1998). Activation of cPKC', regardless of the stimulus, seemingly involves translocation from the cytosol to specific binding domains at cell membranes and can involve four basic interactions. First, these isoforms can form catalytically inactive, membrane-associated complexes in a Ca2+-dependent manner; second, they may form two membrane contacts in the C1 and C2 domains (see legend to Figure 1 for description of PKC domain sequence); third, membrane interactions can occur that facilitate conformational changes of the PKC molecule, which are distinct from an activation event; and fourth, additional changes can occur following DAG/phorbol diester binding to the membrane-associated kinase. See Zidovetzki & Lester, 1992; Lester et al., 1990; Bazzi & Nelsestuan 1987 for further details. The activity of PKC is controlled by its compartmentalization within the cell. Mochly-Rosen (1995) indicated that specific anchoring proteins (immobilized at particular intracellular sites) localize the kinases to their sites of action. These include 'receptors for activated C-kinase' (RACKS), annexins and other cytoskeletal proteins. For a protein to be considered a RACK, certain criteria must be fulfilled: (i) it should bind PKC in the presence of activators; (ii) PKC binding to recombinant RACK1 should not be inhibited by a pseudosubstrate peptide; (iii) binding of PKC to RACK1 should be saturable and specific; and (iv) RACKS should contain a sequence homologous to a PKC binding motif. Following an increase in [Ca2+]i, PKC interacts with the cell membrane in an inactive, but conformationally distinct, form (Bell & Burns, 1991; Huang, 1989). With respect to cPKC' and nPKC', the availability of DAG is essential for their activation through its ability to facilitate the penetration of these isoenzymes into the cell membrane (Lester et al., 1990). Using synthetic lipid bilayers, it has been shown that the region in PKC required for penetration contains cysteines and one or more primary amines. This sequence may have homology with a region in phospholipase A2 that acts as a phospholipid-binding site for the enzyme (Lester et al., 1990). The effects of different phorbol diesters on the activation of PKC' α, β1, γ, δ and ε have been investigated (Ryves et al., 1991) in an attempt to identify isoenzyme-selective activators without success. Activation of cPKC' is critically dependent [Ca2+]i, as well as PS. Indeed, the level of Ca2+ under resting conditions is insufficient to activate PKC. Upon receptor stimulation, and subsequent Ca2+ mobilization, the [Ca2+]i increases transiently and of sufficient magnitude to promote translocation of inactive PKC from the cytosol to the plasma membrane for attachment and activation (Zidovetzki & Lester, 1992). When attached to the membrane, the affinity of PKC for Ca2+ is increased (Nishizuka, 1988) such that activation of the enzyme is achieved. Phosphatidylserine has been shown to bind electrostatically to peptides bearing sequence homology to the pseudosubstrate binding site in PKC (Mosier & McLaughlin, 1991). Since unsaturated fatty acid chain length in DAG and PS affect their potential action as PKC cofactors (Lapetina et al., 1985), hydrophobic membrane-interactions must also apply in the activation process (Brumfeld & Lester, 1990). Other membrane phospholipids may ultimately link extracellular signals to intracellular events through PKC (Nishizuka, 1992). cis-Unsaturated fatty acids such as arachidonic, linoleic and oleic acid can activate PKC in the absence of DAG and PS (Hansson et al., 1986), suggesting an activation mechanism that differs from that effected by DAG. Phosphatidylcholine only activates PKC if a cis-fatty acid is present (Chen et al., 1992), even in the presence of DAG. This is the case for all types of PKC. It has been suggested that fatty acids exert their effect at the regulatory domain of PKC by altering pseudosubstrate binding and unblocking the catalytic site, thereby activating the kinase (Chauhan et al., 1990). It is well known that some, if not all, PKC isoforms can be proteolytically cleaved at the V3 region by the calcium-activated protease, calpain (Kishimoto et al., 1983), to generate a cofactor-independent free catalytic subunit known as protein kinase M (PKM). Calpain exists as two distinct isoforms that have a low (μ-calpain) and high (m-calpain) requirement for Ca2+ respectively, where m and μ reflect millimolar and micromolar [Ca2+] respectively (Michetti et al., 1995). The physiological relevance of the 'calpain-product' was not understood at the time of its discovery but it should not be regarded as an 'unregulated' enzyme since its generation is, in fact, regulated by proteolysis (Shea et al., 1994). A Ca2+/phospholipid-independent kinase similar to PKM has been observed in several tissues such as the neutrophil (Melloni et al., 1986). In that cell, the rate of disappearance of Ca2+/phospholipid-dependent PKC activity correlated with the appearance of a 65 kDa Ca2+/phospholipid-independent kinase that was identical to that reported for proteolytically modified PKC. Thus, the proteolytic cleavage of the membrane-bound protein can be considered an alternative mechanism of activation. Prolonged exposure of mammalian cells and tissues to phorbol diesters and bryostatins has been shown to desensitize agonist- and phorbol-diester-induced responses by a mechanism that can involve down-regulation of conventional and novel PKC isoforms (Newton, 1995; Smith et al., 1985; Huwiler et al., 1994). Although calpains have been implicated in this down-regulation (Eto et al., 1995a; Kishimoto et al., 1989), the evidence is not convincing as it derives from studies with small molecule inhibitors of limited specificity (see Lee et al., 1997). It seems likely that increased vesicle trafficking including lysosomal endocytosis may account for down-regulation of some PKC species (Goode et al., 1994; 1995; Junco et al., 1994). Indeed, isoform-specific PKC down-regulation has been observed in Schizosaccharomyces pombe in response to phorbol diesters (Goode et al., 1995). Alternatively, activation of the ubiquitin/proteasome pathway may play a dominant role in the down-regulation of certain PKC isoforms including PKCα and PKCε (Lee et al., 1996; 1997). All PKCs except the δ isoform express, so-called, PEST sequences (hydrophilic polypeptide segments enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)), that target proteins for degradation by the proteasome (Rechsteiner & Rogers, 1996). An additional level of complexity has been the finding that dephosphorylation of activated PKCs predisposes them to ubiquitination (Lee et al., 1997). PKC isolated from many tissues is subject to oxidation by a number of agents including peroxide, N-chlorosuccinimide and periodate. In all cases, oxidation is dependent on the presence of Fe2+ and yields a modified form of the enzyme that is constitutively active. Thus, in the case of cPKC', Ca2+ and phospholipid are not required for activity (Palumbo et al., 1992; Gopalakrishna & Anderson, 1987; 1989; Allen et al., 1994; Webb et al., 1997a). Oxidative inactivation of PKC by hydrogen peroxide (H2O2) is enhanced by Ca2+ or by phorbol diesters in the absence of Ca2+. Gopalakrishna et al. (1989) demonstrated that ATP protects the catalytic site from oxidation indicating that the ATP-binding domain features redox-sensitive residues. Under this condition, oxidants increase cofactor-independent PKC activity by exerting an affect at the regulatory site. Using rat brain PKC, it was also demonstrated that mild oxidation of PKC increases Ca2+/phospholipid-independent activity whereas further oxidation inactivates the enzyme (Allen et al., 1994). N-Chlorosuccinimide as well as H2O2 down-regulate phorbol diester binding to PKC (Gopalakrishna et al., 1987) indicating that oxidation of the DAG-binding domain markedly reduces the affinity of phorbol diesters and DAG for oxidized PKC. This inactivation was shown to be more rapid for membrane-associated PKC than the cytosolic form. At micromolar concentrations, periodate activates PKC through oxidation at the regulatory site, whereas at higher concentrations of the oxidant the enzyme is inactivated through an action at the catalytic domain (Gopalakrishna et al., 1991). Thus, selective regulatory site oxidation constitutively activates PKC whereas oxidation at the regulatory and/or catalytic domain, inactivates the enzyme irreversibly. We have shown (Webb et al., 1997a) that constitutively active cPKC isoforms exist in bovine ASM and behave as 'unregulated' kinases due to selective oxidation of the regulatory domain. In this respect, it is interesting that treatment of smooth muscle cells with phorbol diesters increases the concentration of intracellular reactive oxygen species (MietusSnyder et al., 1997) raising the potential for exaggerated PKC-driven responses. Mechanistically, oxidation of PKC may represent a unique method of regulation. Just as the apparently 'unregulated' activity of PKM is, in fact, controlled by proteolysis, so is the 'unregulated' activity of oxidized PKC at the regulatory site. Indeed, the constitutive activity is abolished by further oxidation through inactivation of the enzyme at the catalytic core. It is possible that activation of PKC may occur under conditions of oxidative stress, typified by inflammatory disorders such as chronic obstructive pulmonary disease, asthma and arthritis. Protein kinase C contains certain thiol residues that may serve to regulate the enzymes' activity by acting as substrates for S-nitrosylation. Gopalakrishna et al. (1993; 1995) demonstrated (using a number of nitric oxide-generating agents such as sodium nitroprusside) irreversible inactivation of PKC activity and a loss of phorbol diester binding due to disulphide bridge formation following oxidation of the nitrosylated thiols. Inhibition of calpain by S-nitrosylation also may represent an indirect process through which PKC is regulated (Michetti et al., 1995). Until recently, there have been few published studies to suggest that PKC is phosphorylated by kinases other than itself. However, it is now appreciated that PKC isoforms are phosphorylated at an 'activation loop' C-terminal serine/threonine residue in the sequence TFCGTP by phosphatidylinositol-trisphosphate-dependent kinase (PDK1). This is followed by an additional phosphorylation (probably PDK-mediated) or autophosphorylation at a more C-terminal serine/threonine residue in the sequence FSY/FTY. Nineteen amino-acids N-terminal to this FSY/FTY phosphorylation motif is another autophosphorylation site (the 'so-called' TP site) (Bornancin & Parker, 1996; 1997; Stempka et al., 1997; Li et al., 1997). The order of these final two phosphorylation events is not elucidated, though it is clear that each event induces necessary conformational changes in the PKC molecule that result in altered thermal stability, resistance to phosphatases and catalytic activity (Stempka et al., 1997; Newton & Koshland, 1990; Bornancin & Parker, 1996; 1997). Autophosphorylation of PKC requires great flexibility of the protein structure, and the region surrounding these sites of phosphorylation contains no common recognition sequence or similarity with substrate phosphorylation sites (Flint et al., 1990). A number of features of PKC autophosphorylation are evident. In particular, it requires higher concentrations of PS than are required for phosphorylation of exogenous substrates (Bazzi & Nelsestuan, 1992; Newton & Koshland, 1990) due to different PS requirements of aggregation by divalent cations. The expression and distribution of PKC isoforms varies markedly between cells and tissues (Nishizuka, 1988). Some isoforms (e.g. PKCα) are ubiquitously expressed whereas others seem to be restricted to certain tissues (see Table 1). In ASM, similar patterns of expression of PKC isoforms have been found in the larger central airways of several species (Table 2). In bovine trachealis there is expression of conventional α-, βI-, and βII-PKC isoforms as well as the novel δ-, ε and θ-PKC variants together with trace amounts of atypical PKCζ (Webb et al., 1997a). Canine trachealis shows a similar profile, except that PKCα is apparently absent (Donnelly et al., 1995). With the availability of monoclonal antibodies to additional members of the aPKCs, a recent investigation also identified PKCμ and -ι/λ isoforms in porcine trachealis (Togashi et al., 1997). In human trachealis, there is expression of the conventional α, βI, and βII PKC isoforms as well as novel (δ, ε, ζ, θ) and atypical (ζ) variants (Webb et al., 1997b). Where discrepancies between species have been noted in expression of particular isoforms, it is possible that this involves failure of antibodies raised against human epitopes to cross-react with those of different species, or to the presence of contaminating non-muscle cell types in the tissue preparations. PKCγ, often abundantly expressed in neuronal tissues, has not been detected in ASM from any species thus far investigated. Our present understanding of smooth muscle contraction is based on the studies of & and & Using muscle these two groups of independently the which that contraction of muscle is the result of a relative between two of protein and with the formation of studies have been to the and regulation of smooth muscle contraction (Allen et al., 1994; & 1994) and have been discovered between the processes that generation and et al., et al., & 1992). An understanding of smooth muscle contraction and such as PKC, are requires of the structure of the proteins and they with each other ultimately to effect is a protein that in muscle it is of a single of and two of light myosin is composed of a and a The of many myosin to form the of each myosin the which has a the binding site for attachment to and two domains of catalytic activity that the required for Both of light are also located at the of myosin and it is those of kDa that are to exert regulatory the interaction (see is the other In as or exists as that in cells into or In two First, each subunit of the chain features a binding site for the of which the and of between the by a process activates myosin when are phosphorylated by a and enzyme known as myosin light chain kinase (MLCK) & 1977; & 1977; et al., Thus, in the absence of Ca2+, is catalytically However, when [Ca2+]i increases some the resting Ca2+ bind to one or more hydrophobic domains for which has high and a is phosphorylation to activate myosin which the ATP required for the formation of (Figure 2). of generation and in ASM PKC and When a acting through a receptor is to an ASM cell of the there a increase in the [Ca2+]i of sufficient magnitude to all four Ca2+-binding sites on In this form is to active myosin light chain kinase which phosphorylates the kDa light of myosin activation of myosin and, in When the [Ca2+]i the resting Ca2+ from and is by myosin light chain However, in many cases, is for generation is the activation of a novel protein kinase C (PKC) isoform such as PKCε by diacylglycerol which is also by the and phosphorylates an protein, Under resting conditions the ability of to activate myosin and so the muscle However, in the phosphorylated from thereby its effect on myosin contraction due to the phosphorylation of by the resting activity of is thought to be regulated by phosphorylation and protein has been shown to to a form that myosin activity (see for further Although phosphorylation for generation in ASM, evidence exists that a different as well as or in to may be for This was based on that is when are and myosin activity is and to the of a modified or between and myosin which was the et al., & However, subsequent studies identified with the (see & for and et al. to as a by phosphorylation when high are with contraction and ATP the general finding that ATP and [Ca2+]i in ASM are low and that phosphorylation can be from myosin activity has to the that is controlled and/or at in by a different is that the of are in some modified to a which is for in this is that phosphorylation is necessary and sufficient to explain this modified interaction and represents so-called, Another that has much and is not with the is that is due to an that may involve the of other proteins including and/or one or more of the Both are including the potential role by PKC. These are diesters are as activators of conventional and novel PKCs due to their ability to for and & this to if is a process that is by PKC through the phosphorylation of Using porcine smooth muscle these reported that the phorbol phorbol a increase in that was by Ca2+ and that phorbol diesters can promote Ca2+ into smooth muscle (see & that the contraction was by Ca2+ through their ability to promote the phosphorylation of and with this is the finding that although PKC the phosphorylation of on several this is not with an increase in myosin activity et al., 1987; et al., and co-workers thus that phosphorylation account for the effect of without the to involve an of this is by et al. (1989) observed that the phosphorylation of at and in smooth the of which was phosphorylated by but not PKC. The that phosphorylation is Ca2+-dependent that this response through the activation of with much of the Ca2+ through Ca2+ the types of thus far it is the or that is most in the plasma membrane of smooth muscle cells including ASM & 1994). The of and as has to the discovery that are large proteins with in to form an In the the subunit is as a target for In particular, it is phosphorylated by PKC together with the subunit (also a substrate for results in activation by an by the N-terminus of et al., 1998). such regulation is by PKC in smooth muscle is but some indirect evidence to this is In ASM from the & et al., 1993), et al., 1986), & 1989), rat et al., 1994) and & 1996; et al., low concentrations of phorbol diesters similar to those in the (see is by inhibitors of PKC, including and et al., as well as by & 1995). The rate and magnitude of generation is enhanced by Ca2+ and of such as & 1985; & 1996). of extracellular Ca2+ or of and phorbol & 1985; et al., these suggest that phorbol diesters promote generation in ASM, at in by a process that involves an increase in the of by phosphorylation of the Ca2+ phosphorylation and