Categories of protein kinase activity

Protein kinases can be divided into two broad classes on the basis of whether the amino acids serine/threonine or tyrosine are the primary phosphate acceptor sites. The following sections will consider first the serine/threonine-directed enzymes, subdivided on the basis of stimulus sensitivity, and second, the tyrosine-directed enzymes, either as receptor-linked or nonreceptor-linked.

Serine/threonine-directed protein kinases

Cyclic nucleotide-dependent protein kinases The first stimulus-sensitive protein kinases to be studied were those regulated by cAMP. This second messenger is generated by adenylyl cyclase which couples to multiple cell surface receptors through heterotrimeric GTP-binding proteins. Cyclic nucleotide levels are also subject to control through the action of phosphodiesterases. cAMP-dependent protein kinases (PKAs) consist of two subunits: a catalytic subunit and a regulatory subunit. The catalytic subunit is normally inactive when complexed with the regulatory subunit. Interaction of the complex with cAMP results in subunit dissociation and subsequent activation of the enzyme. Multiple forms of each sub-unit class have been characterized. Substrate specificity of PKAs is quite broad and the response to elevated cAMP exhibits a diverse array of downstream consequences.

Ca2+/calmodulin-dependent protein kinases There are multiple protein kinase activities that are controlled by changes in intracellular Ca2+ levels through interaction with calmodulin, a Ca2+-binding regulatory protein. When Ca2+ levels are elevated, Ca21-calmodulin complexes associate with several different protein kinases and such association results in enhanced catalytic activity of the enzymes. At least six different forms of calmodulin-dependent protein kinases have been defined. Three of these isoforms (phosphorylase kinase, myosin light chain kinase, and Ca21 /calmodulin kinase III) exhibit a high degree of substrate specificity. Isoforms I, II and IV exhibit much broader specificity and are referred to as multifunctional forms. The different forms exhibit substantial structural complexity, frequently being composed of multiple distinct subunits.

Phospholipid-dependent protein kinases Phospholipid hydrolysis is now a well-documented early event in intracellular signaling. The activation of different phospholipase activities with highly distinct substrate specificities is linked to multiple receptor systems in using a variety of distinct molecular coupling mechanisms.

There are two broad categories of protein kinase activities that are sensitive to specific phospholipid hydrolysis products which act as second messengers. The first of these families is a group of enzymes known collectively as protein kinase C (PKC). These enzyme activities were discovered as a Ca2 and phospholipid-dependent activity and were subsequently found to be the target of the tumor promoter phorbol diesters. At least 11 structurally distinct forms of PKC-like enzymes are known and these can be divided into three subgroups on the basis of cofactor requirements. These include a, f> and y isoforms which require Ca2 + , phospholipid, and a diacylglycerol (DAG) activator, the 8, e, ^ and a isoforms which are Ca2 -independent but still require DAG, and the £ and \ isoforms which require only phospholipid for activity. In addition, two structurally related enzymes known as protein kinase D and PKC|x are also DAG-sensitive and Ca2' -independent.

Activity of the Ca24-dependent isoforms of PKC may require the concomitant elevation of intracellular Ca2'. These cofactor and activator requirements provide some opportunity for stimulus-dependent specificity in the function of the different isoforms of PKC. However, there appears to be little substrate specificity inherent in PKC structure and, at least in vitro, many proteins can serve as targets for the different forms of enzyme. One potentially important determinant of specificity may be subcellular localization. Some forms of PKC show preference for association with different membrane sites and may so associate in an activation-dependent fashion. Furthermore, PKC isoforms exhibit interaction with other nonprotein kinase proteins which may also be important determinants of selective function or activation. Following activation, some forms of PKC undergo Ca2~-dependent proteolysis. Cleavage may selectively remove the regulatory domain of the protein, rendering it constitutively active at least transiently.

A second form of phospholipid-dependent protein kinase has been identified whose activity is modulated by ceramide, a hydrolysis product of sphingo myelin. Several different extracellular stimuli (tumor necrosis factor a (TNFa), interleukin 1 (1L-1) and vitamin D?) have been convincingly linked with the sequential activation of a neutral spingomyelinase which generates ceramide, and at least some downstream signaling events are mediated by activation of the ceramide-dependent protein kinase. The enzyme is a member of a large family of serine/threonine-directed protein kinases which recognize substrate peptides in which the phosphoacceptor site is N-ter-minal to a proline residue. While specific substrates are nor definitively known, activation of the ceram-ide-dependent signaling path way (s) has been linked with the activation of the MAPK cascades, with activation of the NFkB transcription factor, and with the stimulation of apoptosis.

The MAPK signaling pathway A major functional class of protein kinase activities which has been studied intensively in recent years is now known collectively as the mitogen-activated protein kinase (MAPK) cascade. While there are multiple structural forms of protein kinase activity which participate in the MAPK cascade, they are linked together in a functional unit which can mediate information transfer from the exterior of the cell to the nucleus, resulting in stimulus-dependent alterations in gene expression. Indeed these signaling pathways can be initiated by a highly diverse array of important extracellular stimuli which induce both cell proliferation and functional differentiation.

Two general features of this signaling process deserve special comment. First, there are multiple forms of the MAPK cascade that may coexist in the same cell and which exhibit differential sensitivity to different forms of extracellular stimulation. Second, the different MAPK cascades exhibit at least five sequential layers of protein kinases. At each layer, multiple isoforms of kinase activity have been described. The nomenclature for these enzymes is quite complex and the simplest system relates all kinase activities to the originally identified mitogen-acti-vated protein kinase termed MAPK. Thus the MAPK kinase is termed MAPKK while the next upstream enzyme is termed MAPKKK or MAP3K and so on. Enzymes which are downstream of MAPK are termed MAPK-activated protein kinases or MAPK-APK.

The MAPKs are members of the proline-directed protein kinase family (like ceramide-activated protein kinase, see above). MAPKKs are relatively unique enzymes which phosphorylate MAPK on both tyrosine and serine or threonine residues, both events being required for full activation of MAPK activity. The MAPK cascades can be initiated through multiple entry points and include action of receptor-linked protein tyrosine kinase activity (see below), phospholipid-dependent kinases, and intracellular receptor-initiated events (steroids). Furthermore, there is substantial cross-talk between the distinct cascades. Through such combinatorial mechanisms this system is capable of generating remarkable diversity in information transfer.

Other serine/threonine protein kinases There are no doubt many additional serine/ threonine-directed protein kinases which participate in stimulus-response coupling. Prominent examples include the double-stranded RNA-dependent protein kinase known as PKR which is associated with response to interferon (IFN) and with acquisition of antiviral function. Recent studies using mice in which this enzyme has been deleted by gene targeting through homologous recombination indicate that this enzyme may play an important role in cellular response to extracellular stimulus.

Another class of enzymes known to exhibit change in activity in response to stimulation are collectively known as casein kinases based upon their preference for casein as a substrate. Two classes of casein kinases have been studied. Both show relatively broad substrate specificity. Although casein kinase activities vary in response to extracellular stimulation, there is little mechanistic understanding of how enzyme activity changes during cell treatments nor how important these enzyme forms may be in processing such extracellular information.

Tyrosine-directed protein kinases (PTKs)

Protein kinases that utilize tyrosine residues as the principal phosphate acceptor site have been classified into two categories on the basis of whether the enzyme activity is an integral part of a cellular receptor or has no demonstrable receptor function.

Receptor-linked PTKs Although the first tyrosine-directed protein kinase discovered was not receptor associated, the linkage of tyrosine-directed phosphorylation with response to extracellular stimulation focused early attention on those enzyme activities which were integral parts of the receptor protein structure. This paradigm is now well-established and is associated most closely with receptors for growth factors such as insulin and insulin-like growth factors, epidermal growth factor, fibroblast growth factors and the macrophage colony-stimulating factor CSF-1. The linkage to cell proliferation is not exclusive as these stimuli are also capable of producing developmental changes in cells related to the acquisition of selective functions. Receptor-linked PTKs are activated upon ligand binding by receptor sub-unit aggregation. Activated enzymes phosphorylate the receptor itself as well as other substrates, at least some of which associate with the receptor via protein domains which recognize and bind to phosphotyros-ine residues. Thus the phosphorylation induced by ligand-receptor interaction initiates a signaling cascade which may involve additional protein kinases as well as other enzymes. The receptor-linked PTKs are highly pleiotropic and can couple to many downstream signaling pathways, including those involving the serine/threonine protein kinases discussed above.

Nonreceptor-linked PTKs There are at least eight categories of PTK that are not part of any stimulus-binding protein and exhibit no extracellular domain. These eight subclasses are distinguished by specific structural features. All possess at least one kinase domain which contains the active site. Many possess regions that allow interaction with phosphotyrosine residues or with other specific amino acid sequence motifs which are expressed on the cytoplasmic domains of specific receptors. In addition, enzyme activity is frequently subject to positive or negative regulation by phosphorylation on critical tyrosine residues. These latter two characteristics are critical to the function of at least three classes of nonreceptor PTKs. Cell surface receptors aggregate upon ligand binding, which results in the activation of PTKs associated with the cytoplasmic domains of the receptors. These enzymes phosphorylate themselves as well as the receptor subunits, leading to interaction with a multitude of downstream signaling intermediates in a fashion remarkably similar to that described above for the receptor-linked PTKs.

Nonreceptor PTK classes that function in this manner include the Janus family (JAK1, JAK2, JAK3, Tyk2), the Syk family (Syk, Zap), and the Src family (Src, Yes, Fyn, Yyn, Lck, Blk, Hck, Fgr, and Yrk). Many of these mediate signaling pathways restricted to hematopoietic cell types. Although the other classes of nonreceptor-linked PTKs have not been so closely associated with receptor-mediated events, they are also likely to be involved with intracellular communication and regulation.

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