Active and Inactive Protein Kinases: Structural Basis for Regulation

lytic groups and relief of steric blocking to allow access Protein kinases and phosphatases play pivotal roles in of substrates to the catalytic site. The activation segregulating and coordinating aspects of metabolism, ment and the control of its conformation via phosphorygene expression, cell growth, cell motility, cell differentilation plays a key role in these transformations. It can ation, and cell division. As a result, if cellular life is to be involved in recognition of regulatory subunits, in aufunction in an orderly manner, the switching on and off toinhibition of substrate binding, and in promotion of of protein kinases and phosphatases is as crucial for the correct orientation of domains and catalytic site resitheir function as their catalytic activity. dues. This review summarizes our current understandThe total number of distinct kinase domain sequences ing of control by the activation segment based on recent available is now approaching 400 (Hardie and Hanks, structure determinations of active and inactive kinases. 1995). Multiple sequence alignments have indicated that The first observation of Thr-197 phosphorylation in all protein kinases should have similar structures, and the activation segment of cAPK was reported in 1979 this has been confirmed by recent crystal structure de(Shoji et al., 1979). Although itwas speculated that phosterminations. Conserved features have been identified phorylation at discrete sites might be of physiological in 12 subdomain regions of all protein kinases, and resiimportance in the regulation of enzyme activity, it was dues from these subdomains have been implicated in not until 1990 that mutagenesis studies indicated the essential roles in enzyme structure and function. Protein significance of this site for the recognition of the regulakinases exhibit variability in other parts of the kinase tory subunit (Levin and Zoller, 1990), and not until 1993 domain, and different kinases may contain additional that it was definitively shown that phosphorylation is domains, additional subunits, or both. These features promoted by an autocatalytic event that is crucial for allow several different mechanisms for control. activation (Steinberg et al., 1993). The crystal structure Control mechanisms that have been recognized to determination of cAPK in 1991 (Knighton et al., 1991a, date include the following: control by additional subunits 1991b) showed the structural importance of Thr-197 or domains that may function in response to second phosphorylation and demonstrated possible roles of messengers (e.g., cyclic AMP binding to the regulatory phosphorylation in promotion of activation. The strucsubunit of cyclic AMP–dependent protein kinase [cAPK], ture provided a definitive model to which other kinases Ca/calmodulin binding to calmodulin-dependent could be related. Also in 1991, both the fission yeast protein kinases, and Ca and diacyl glyerol binding to cell division control kinase (cdc2) (Gould et al., 1991) N-terminal domains of protein kinase C); control by addiand the microtubule-associated protein kinase (MAPK) tional subunits whose level of expression varies de(Payne et al., 1991) were found to be activated by phospending on the functional state of the cell (e.g., cyclin phorylation on residues that mapped to a position simiregulation of the cyclin-dependent protein kinases lar to Thr-197 in cAPK. These results showed the impor[CDKs]); control by additional domains that target the tance of this site not only as an autophosphorylation kinase to different molecules or subcellular localizations site, as in cAPK, but also as a site involved in kinase (e.g., the SH2 and SH3 domains of the Src kinases); cascade activation mechanisms. For the tyrosine kicontrol by additional domains that inhibit the kinase nases, autophosphorylation of pp60 at position Tyractivity by an autoregulatory process (e.g., myosin light 416 (now known to be in the activation segment) had chain kinase [MLCK]); and control by phosphorylation been shown in the early 1980s, and its significance for and dephosphorylation by kinases and phosphatases. control in the cellular counterpart of Src kinase was Phosphorylation of specific threonine, serine, or tyrosine established by 1987 (reviewed by Hunter, 1987). The residues may occur at a number of sites. Some of these following year, trans-autophosphorylation of the insulin are located in the N-terminal or C-terminal portions of receptor tyrosine kinase (IRK) was elaborated, and the the polypeptide chain, which lie outside the kinase dosimilarity in sequence location of some of these sites main (e.g., in Src kinase and calmodulin-dependent kito that in Src kinase and its relatives and in cAPK was nase II [CaMKII]) or on othersubunits (e.g., in phosphorynoted (reviewed by White and Kahn, 1994). As more and lase kinase [PhK]). A key aspect of regulation recognized more kinases have been discovered and sequenced and in recent years is that many protein kinases are phosfurther kinase cascades established, it is recognized phorylated on a residue(s) located in a particular segthat control by phosphorylation in the activation segment in the center of the kinase domain, which is termed ment is a property of most, but not all, protein kinases

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