Structural framework for the protein kinase family.

Further

ANNUAL REVIEWS

Quick links to online content Annu.

Rev. Cell Bioi. 1992. 8:429--f>2 Copyright © I992 by Annual Reviews Inc.

All rights

reserved

Annu. Rev. Cell. Biol. 1992.8:429-462. Downloaded from www.annualreviews.org by Johns Hopkins University on 10/10/13. For personal use only.

STRUCTURAL FRAMEWORK FOR THE PROTEIN KINASE FAMILY l l2 l Susan S. Tay,lor , Daniel R. Knighton , , Jianhua Zheng , Lynn 23 l 4 F. Ten Eyck and Janusz M. Sowadski , ,

,

University of California, San Diego, 9500 Gilman Drive, La 10lla, California 92093-0654

KEY WORDS: protein phosphorylation/dephosphorylation. cAMP-dependent protein kinase, pro tein kinase homologies, protein kinase regulation, enzyme structure/function,

phosphotransferases

CONTENTS PROTEIN PHOSPHORYLATION HistoricalOverview .............................................. .

430 430 432

Diversity in the Protein Kinase Family ............................... .

.

cAMP-DEPENDENT PROTEIN KINASE .. . . . ... . ... . . . . . ..... . .. ... . . . .

.

433

.

434 434 435 437 437 439

CRYSTAL STRUCTURE OF THE CATALYTIC SUBUNIT: A Walk Through the Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

General Topology ........ . ..... . . ................................ . A m ino Terminus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ATP-Binding Domain ........... . ................................. . Peptide-Binding Domain . . . . . . . . . . . . . .. . .. . . .. . .. . .. . . . . .. . . . . . .. . . . A utophosp horylation Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

THE CATALYTIC SUBUNIT IS A STRUCTURAL FRAMEWORK FOR THE PROTEIN KINASE FAMILY . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

Boundaries of the Catalytic Core .................................... . Invariant Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conserved Sequence Motifs ......... . ............................... . General Sequence Similarities Predict Conserved Secondary Structure . . . . . . . . . . Location of Inserts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary ........ ' " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

440 441 442 443 449 449 450

Department of Chemistry Department of Medicine Department of Biology San Diego Supercomputer Center, P.O. Box 856 08, San Diego, California 92188-9784

429

0743-4634/92/1115-0429$02.00

430

TAYLOR ET AL

SOME COMMON THEMES FOR REGULATION OF PROTEIN KINASE ACTIVITY . .................. ... . Occupancy of Peptide-Binding Sites with an Inhibitory Peptide-Like Segment ...................................

451

Role of Phosphorylation ... ... ............ . ..... ....... .............

451 454

SUMMARY ............................. . ................... . .....

456

PROTEIN PHOSPHORYLATION


Historical Overview Protein phosphorylation was first elucidated as a mechanism for covalently and reversibly regulating enzyme activity in the 1 950's. This regulatory mechanism emerged from several laboratories studying the breakdown and synthesis of glycogen in the liver (Fischer & Krebs 1955; Sutherland & Wosilait 1 955). At that time, and in fact for over two decades, the full magnitude of protein phosphorylation as a regulatory mechanism in eukaryotic cells was not fully appreciated. In the late 1970's it became apparent that not only were there a large number of protein kinases, but also that these enzymes played key regulatory roles in many cellular processes such as hormone responsiveness, cell growth and division (Lorincz & Reed 1 984) , and gene expression (Yamamoto et al 1 988; Karin 1 990; Foulkes et al 1 99 1 ) . The discovery in 1 979 that the transforming protein of Rous sarcoma virus, pp60v-src, had intrinsic protein kinase activity (Collett & Erickson 1 978) and, subsequently, that pp60src transferred phosphate to Tyr rather than Ser or Thr (Hunter & Sefton 1 980), opened up a major new dimension to the field of protein phosphorylation. Almost all processes that involve receiving an extracellular signal and then translating and amplifying that signal inside the cell involve protein phosphorylation at some critical step. This is a highly evolved and sophisticated process that is critical for nearly all aspects of eukaryotic regulation. Defective protein kinases also are capable of transform ing normal cells into malignant cells (Bishop 1 987) . A s more protein kinases were recognized i n the 1 980s, it became clear that this was a diverse family. The first obvious diversity was in peptide recognition, not only for the phosphate acceptor group, but also for the sequences t1anking the phosphorylation site . Two general classes emerged: those protein kinases that transfer phosphate to Ser or Thr and those proteins that transfer phosphate to Tyr (Krebs & Beavo 1979). A few protein kinases, such as wee 1, now appear to be capable of phosphorylating both Ser/Thr and Tyr (Lindberg et al 1 992) . The protein kinases are also structurally widely diverse in terms of size, subunit structure, and subcellular localization . A final area of diversity is in the mechanism of activation. With the exception of the oncogenic enzymes , all protein kinases are themselves tightly regulated enzymes that are switched on or off, either directly or indirectly, in response


PROTEIN KINASE STRUCTURE

431

2+ to specific signals as diverse as a growth hormone, cAMP, Ca , or a tumor promoter. Thus we have a diverse family of enzymes critical for cell growth and development and essential for the cell to communicate with its extracel lular environment. In spite of this considerable diversity, it also was apparent that the protein kinases were evolutionarily related (Barker & Dayhoff 1982) and have evolved, in part, divergently from a common origin (Hanks et al 1988). Specifically, all of the eukaryotic protein kinases share a conserved catalytic COfe of approximately 260 residues, shown in Figure 1, which indicates that all have conserved a common mechanism for catalysis . Indeed , those residues important for catalysis and for MgATP binding are generally conserved throughout the entire protein kinase family. This review briefly summarizes some of the key features that exemplify the structural diversity of the protein kinase family and then focuses on the crystal structure of the catalytic subunit of cAMP-dependent protein kinase with particular emphasis on how this relatively simple protein kinase structure can serve as a framework for the entire family of enzymes .

.: .:

mi

iii:

I I

iii;

U

;U iii:

mi Ii

��

��

iii:

iii:

: : : : ; : ; ; ;

I

cAPK cGPK

I

PKe

I

MLCK

S)..� �

I

Phos Kin cde2 c-src v-erbB EGF Receptor

I I

Figure 1 Structural diversities and similarities in the protein kinase family. A comparison of amino acid sequences identifies conserved and variable regions in various protein kinases. The conserved catalytic core is shown in black. The catalytic subunit of cAPK contains 350 amino acids, and the core extends from residue 40 to residue 300. Regions thought to be important for regulation are cross-hatched. The putative membrane-spanning segment in the EGF receptor is stippled. Three conserved sequence motifs corresponding to the glycine-rich loop (small black

rectangle), Lys72 (circle), and Aspl84 (square) are indicated as well as N-terminal myristoylation (m). Thc upper panel shows kinases specific for Scr/Thr; the lower panel shows kinases specific for Tyr. Alignments are based on the following sequences: catalytic subunit of CAMP-dependent protein kinase (cAPK) (Shoji et al 1983); cGMP-dependent protein kinase (cGPK) (Takio et al 1 984b); protein kinase C (PKC) (Parker et al 1 986; Coussens et al 1980; Knopf et al 1 986); myosin light chain kinase (MLCK) (Takio et al 1985); phosphorylase kinase (Phos Kin) (Reimann et al 1984); cdc 2 (Hindley & Phear 1984); c-src (Czemilofsky et al 1980;

Martinez et al 1 9R7); epidennal growth factor receptor (EGFR) (Ullrich et al 1984); v-erbB (Yamamoto et al 1983).

432

TAYLOR ET AL

Diversity in the Protein Kinase Family A brief description of just a few protein kinases, summarized in Figure 1 , is

sufficient to demonstrate the diversity of this family. Protein kinase C, for

example, is a cytoplasmic

rotein that is transiently associated with mem r branes. It is activated by Ca +, phosphatidylserine, and diacylglycerol as well

as by tumor-promoting phorbol esters. The major regulatory sites that respond

to these various activators lie proximal to the catalytic core (Nishizuka

Bell

& Bums 199 1 ) .

1 988;


The calcium/calmodulin-dependent protein kinases represent another solu

ble and cytosolic subgroup of the protein kinase family, and even within this

group there is great diversity. Unlike PKC, which phosphorylates many

substrates, these Ca2+/calmodulin-dependent enzymes are often specific for

one substrate. Myosin light chain kinase (MLCK) is a monomer (Kemp

& 1 990), while Ca2+ /calmodulin-dependent protein kinase II is a dodecamer (Schulman & Lou 1 989) . Both are typically activated by the binding of Ca2+ Icalmodulin. Glycogen phosphorylase kinase (a4!34'Y484) contains 1 6 Stull

subunits where the ),-subunit is the catalytic component and the 8-subunit is

calmodulin. Activation of phosphorylase kinase can be mediated by phosphor

ylation or by Ca2+ binding to the 8-subunit (Pickett-Gies

& Walsh 1986) . In

all cases, the Ca2+/calmodulin binding site lies distal to the catalytic core.

The cell division-cycle protein, cdc2, is a soluble protein kinase that serves

as a critical switch for entry into mitosis. It is closely related to the family

of mitogen-activated kinases. It is one of the smallest protein kinases and depends on association with another protein, cyelin, for activity (Norbury

Nurse

1991; Draetta 1 990; Freeman & Donoghue 199 1 ) .

&

The growth factor receptors such as the epidermal growth factor (EGF)

receptor, the platelet-derived growth factor (PDGF) receptor, and the insulin

receptor represent quite a different motif. These proteins contain a large extracellular growth factor-binding domain, a single putative membrane-span

ning segment, and an intracellular protein tyrosine kinase domain. Binding of the growth factor to the extracellular domain induces a conformational change that leads to kinase activation (White

1 99 1 ; Ullrich & Schlessinger 1990).

In addition to all of the protein kinases that are essential for normal cell

growth and development, many oncogenes code for protein kinases that are

defective in their capacity to be regulated; they are typically constitutively active (Bishop

1 987; Freeman & Donoghue 1 99 1) . The

src

family of

protein-tyrosine kinases, for example, contains many examples of proto oncogenes that have mutated into oncogenic variants (Cooper

1 990) . The

major transforming protein of Rous sarcoma virus, pp60v-src, is permanently

anchored to membranes in part by an N-terminal myristoyl group (Buss et al

1 986; Cross et al 1 984; Kaplan et al 1 990). Sites important for regulation of


433

the proto-oncoprotein, c-src, lie on both sides of the catalytic core (Cooper 1 990; Hunter 1 987a, Kmiecik et aI 1 988). In contrast, v-erbB is an oncogenic homologue of the EGP receptor that lacks the extracellular growth factor binding domain (Downward et al 1984).


cAMP-DEPENDENT PROTEIN KINASE In 1958 the first protein kinase, phosphorylase kinase, was purified and characterized (Krebs et al 1 959) . In 1 968 , the second, cAMP-dependent protein kinase (cAPK), was purified (Walsh et al 1 968). In addition to being one of the first protein kinases to be discovered, cAPK is one of the simplest, and consequently it has served and continues to serve as a prototype for the entire family. Its simplicity is due primarily to its mechanism of activation (Gill & Garren 1 969; Tao et al 1970; Brostrom et al 1970) . In its inactive holoenzyme form, cAPK is a tetramer, containing both regulatory (R) and catalytic (C) subunits. Activation is mediated by cAMP binding with a high affinity to the R-subunit, which promotes dissociation of the complex and the release of the free and active monomeric catalytic subunits (Beebe & Corbin 1 986; Taylor et aI 1 990b). Once released from the tethered holoenzyme state , the catalytic subunit is free to phosphorylate substrate proteins. In its dissociated state, the catalytic subunit is also free to migrate into the nucleus , whereas in its holoenzyme form, the enzyme is sequestered in the cytoplasm (Meinkoth et al 1 990; Adams et al 1 990; Nigg 1 990) . The catalytic subunit contains only 350 amino acids and also is relatively small compared to other protein kinases (Shoji et aI 1 983). In addition to its simplicity, the attractive ness of the catalytic subunit as a prototype for the family is further enhanced because it is relatively abundant and can be easily purified in large quantities (Kinzel & Kubler 1 976) . It is also one of the few protein kinases that can be expressed readily in large quantities and in an active form in E. coli (Slice & Taylor 1 989). These combined factors make this enzyme an ideal choice to study and to serve as a prototype for the family. While mutagenesis has provided a wealth of information regarding func tional sites and essential residues in the various protein kinases, it is cAPK that has repeatedly provided the chemical basis for the identification of important sites and residues (Bramson et al 1 984; Taylor et al 1990a). It was the first protein kinase to be sequenced (Shoji et al 1 983) and was also the first protein kinase in which essential residues were identified by affinity labeling and group specific labeling. Lys72, for example, was shown to be essential for MgATP binding by affinity labeling in cAPK (Zoller et aI 1 98 1 ) . Only later was i t appreciated that this residue i s invariant i n all protein kinases and that mutagenesis of this lysine leads to loss of kinase activity (Kamps et


434

TAYLOR ET AL

al 1 984; Staros et al 1 985; Chen et al 1987; Chou et al 1 987; Ebina et al 1 987) . Lys72 was subsequently shown to crosslink to Asp184, another invariant residue , thus placing these two side chains in close proximity to one another at the active site (Buechler & Taylor 1 988, 1 989). Extensive genetic analysis of the yeast catalytic subunit, TPK 1 , also was carried out. One of the most comprehensive studies utilized a global mutagenesis approach in which all the charged residues were systematically replaced with alanine. This charge-to-Ala mutagenesis is a particularly powerful tool for probing a wide range of phenotypic properties from peptide recognition (Gibbs & Zoller 1 99 1 a) and catalysis (Gibbs & Zoller 1 99 1 b), to interaction sites with the R-subunit (Gibbs et a1 1992). This combined chemical and genetic information provides a fairly detailed skeleton for many of the important structural features of this enzyme (Taylor et al 1 990a) . Recently cAPK again pioneered the way for other protein k inases by becoming the first protein kinase for which a crystal structure is available. The structure solution of the catalytic subunit containing a bound 20 residue inhibitor peptide provides not only a detailed molecular view of cAPK, but also a general template for viewing all members of the protein kinase family (Knighton et al 1 99 1 a,b). This review summarizes the structure of the catalytic subunit of cAPK and then extrapolates to some of the general rules that apply to the larger family of protein kinases.

CRYSTAL STRUCTURE OF THE CATALYTIC SUBUNIT: A Walk Through the Protein General Topology

The structure that was solved is the mouse recombinant catalytic subunit (Slice & Taylor 1989). It is kinetically nearly identical to the mammalian enzyme; however, it lacks a myristoyl moiety at its amino terminus. The enzyme was co-crystallized with the inhibitor peptide shown in Figure 2. The peptide is derived from the naturally occurring heat stable protein kinase inhibitor (PKI) (Walsh et al 1971; Scott et al 1985b) . The small segment at the amino terminus of PKI, PKI(5-24) , contains most of the inhibitory potency (Cheng et al 1986; Scott et al 1985a) . This peptide contains the consensus site generally shared by all inhibitors and substrates of cAPK, while the first 8-10 residues convey the unusual high affinity binding properties associated with PKI (Walsh et al 1990) . The general structure of the catalytic subunit is shown in Figures 3 and 4 . Overall , i t i s a slightly ellipsoid protein with two lobes. The smaller lobe corresponds primarily to the amino terminal segment (residues 1 5- 1 27) and constitutes most of the important features necessary for MgATP binding. The remaining residues constitute most of the large lobe. This large lobe is


High Affinity Site .

Consensus Site . ..

PKI(5-24):

Ki

Kemlide:

Km KI

Ata-Kemplide:

RI-Subunit: R 11- Sub unit:

'O"�C'\:�""':'NJ"""��.;r..:../"o::."'U'��;::;I'\o�",,",,

-11


Figure 2

-6

435

-3 -2

P

=

= =

2.3nM

16flM 300flM

appK appK

=

O.2-0.3nM O.2-0.3nM

+1

•

Substrates and inhibitors that interact with the catalytic subunit of cAPK. The upper

peptide is PKI(5-241 that was co-crystallized with the catalytic subunit. The consensus site (P-3 through P+ 1) is indicated by an arrow. as is the region that conveys the high affinity binding properties of this peptide. Residues most important for PKI(5-24) binding are indicated by a dot (Walsh et al 1990). The substrate and inhibitor heptapeptides and their binding constants are I indicated (Kemp et al 1977; Whitehouse et al 1983). The inhibitor region of the R a-subunit II (Titani et al 1 984) and the R a-subunit (Takio et al 1984a) are also indicated, as well as their Kapp for the catalytic subunit under physiological conditions (Hofmann 1980).

associated with peptide binding and catalysis. Between the two lobes is a cleft, and catalysis occurs in this cleft. Binding of the peptide inhibitor, PKI(5-24), causes a decrease in the radius of gyration, which very likely correlates with conformational changes in this cleft region (Parella et al 1992) . As indicated in Figure 3, MgATP lies at the base of the cleft. Its position was located originally on the basis of difference Fourier maps between the binary and ternary complexes (Knighton et al 1991b). More recently, the structure of a ternary complex containing PKI(5-24) and MgATP was solved, and this provides a detailed profile of the bound nucleotide and the specific contacts it makes with the protein (J. Zheng et al 1 992) . The consensus region of the peptide, shared more-or-less by all substrates and inhibitors of cAP K , occupies the cleft o n the surface o f the large lobe. A dominant feature o f the portion of the peptide that conveys high affinity binding is an amphipathic helix that lies along the surface of the large lobe. From the space-filling model, one can appreciate both the bilobal nature of the molecule and the potential importance of the cleft. The model also shows that the MgATP is quite buried and that recognition of the peptide involves a large area on the surface of the enzyme.

Amino Terminus The first 1 4 residues of the polypeptide chain are not visible in the crystal structure of the recombinant enzyme . In the mammalian enzyme the amino terminal glycine is myristoylated (Carr et al 1 982), and this acyl group is very likely important for stabilizing the protein (Slice & Taylor 1 989; Yonemoto et al 1991). Residue 1 5 begins an a-helix that lies on the surface of the large lobe (Figure 5).


� w 01

� .....::: 5 :;tI � > l'

Figure 3

Space-filling model of the catalytic subunit of cAPK. The catalytic subunit with the substrates deleted

(left)

clearly

emphasizes the cleft between the two lobes. The amino terminal portion of the molecule (residues 1 5-127) is indicated by the light shading. The remainder of the molecule, residues 128-350, is shown in white, and comprises most of the larger lobe, with only the last 20 residues wrapping along the surface of the small lobe. The ternary complex (right) is shown with the peptide, PKI(5-24),

(shaded) and MgATP (black) (J. Zheng et a1 1992; Taylor et aI 1992) (figure reproduced from Taylor et aI, Faraday Discussion No. 93.).


PROTElN KlNASE STRUCTURE

437

Fig ure 4 Alpha-carbon backbone of the catalytic subunit and PKI(5-24). The peptide is indkated by a thin line while the backbune of the catalytic subunit is represented by the thicker line. The peptide, PKI(5-24), is numbered 361 to 380, with residue 377 corresponding to the site of phosphotransfer. The highly conserved residues in the sequence of the protein are also indicated. The position of MgATP is shown based on the refined model of the ternary complex (J. Zheng et aI199 2; Taylor et a1 1 992) (figure reproduced from Taylor et ai, Faraday Discussion No. 93.).

ATP-Binding Domain

The ATP-binding domain begins approximately with residue 40 and extends to residue 127. This domain is dominated by a l3-sheet consisting of five anti-parallel \3-strands. The only region of helix in this domain lies between \3-strands 3 and 4. It is mainly the first three strands plus the C-helix that interact directly with the nucleotide. These specific interactions are discussed below . Peptide-Binding Domain

The larger lobe is associated primarily with peptide binding and catalysis and, in contrast to the ATP-binding domain, is dominated by helices. The single region of \3-structure lies at the surface of the cleft between helix E and F, and most of the residues important for catalysis lie here in the loops that connect these j3-strands . Residues that contribute to peptide recognition are widely dispersed throughout this lobe, beginning with Glu l 27 and extending all the way to Glu33 1 . Approximately 1 4% of the surface area of the enzyme is masked by the binding of this inhibitor peptide (D. R. Knighton et ai, unpublished results).


438

TAYLOR ET AL

Figure 5

Ribbon diagram showing the secondary structure of the catalytic subunit. The chain begins with residue 15 and ends with residue 350. The amino terminal region is shaded. Residue 127 marks the boundary between the ATP-binding domain and the peptide-binding domain. The J3 strands are numbered consecutively while the helices are in alphabetical order (adapted from

Knighton et al 199Ib).

Several residues that are important for peptide recognition by the catalytic subunit were identified well before the structure was solved. Initially the requirements for peptide recognition were mapped using analogues based on known phosphorylation sites (Zetterqvist et al 1 976; Kemp et al 1 977) . The catalytic subunit, in particular, recognizes peptides having the following general consensus sequence: Arg-Arg-X-Ser/Thr-Y, where Y is typically a large hydrophobic residue (Zetterqvist et al 1 990) . This consensus sequence in PKI(5-24) lies along the surface of the cleft with the Ala at the P site facing the 'Y-phosphate of ATP. The two arginines at the P-3 and P-2 positions



439

are anchored firmly by ionic interactions with four glutamates. The P-3 Arg comes close to the region that links the two domains and interacts with both Glu 1 27 and Glu33 1 . The P-2 Arg is fixed by Glu 1 70 and Glu230. Three of these residues were identified by group-specific labeling (Buechler & Taylor 1 988 , 1990). Using charge-to-Ala mutagenesis , Gibbs & Zoller were not only able to identify three of these glutamates in the yeast catalytic subunit, but also were able to correctly predict which arginine each glutamate recognized (Gibbs & Zoller 1 99 1a). The fourth Glu , Glu3 3 1 , is missing in the yeast enzyme. The He at the P+ 1 position is bound in a hydrophobic groove lined by Leu 198, Pr0202, and Leu205 . Hence, the structure is completely consistent with the predicted requirements for recognition of the consensus peptide. The high affinity binding of PKI(5-24) is the result of residues that lie proximal to the consensus site. The P-6 Arg binds to Glu203, and the P- l l Phe binds to a hydrophobic groove and, in particular, is stacked between Tyr235 and Phe239. This P- l l Phe lies on the hydrophobic face of an amphipathic helix . The importance of both the P-6 Arg and the P-l 1 Phe was predicted based on analogue studies (Walsh et al 1990; Glass et al 1 989). The structure of the peptide bound to the surface of the enzyme is very similar to the predicted structure of the peptide in solution based on NMR spectroscopy (Reed et al 1 989; A . Padilla & J. Parello, manuscript in preparation). This helix on the proximal side of the consensus sequence will probably not be a required feature for all protein substrates of cAPK; however, this element of secondary structure may be conserved in some substrate proteins that have an unusually high affinity for the catalytic subunit.

Autophosphorylation Sites The catalytic subunit contains two stable or silent phosphorylation sites, Thr1 97 and Ser338. These sites were identified originally based on amino acid sequencing and were designated as silent phosphorylation sites because the phosphates could not be readily removed with phosphatases (Shoji et al 1 979) . Thrl97, in particular, appears to be a stable part of the folded structure. As seen in Figure 6, three residues, Arg 1 65 , His 87, and Lys 1 89, bind to this phosphate and can easily explain its resistance to hydrolysis by phosphatases. Thrl 97 is fully phosphorylated in vivo when the recombinant enzyme is expressed in E. coli, while an inactive mutant form of the enzyme is not phophorylated, which indicates that the phosphorylations are most likely autocatalytic (Yonemoto et aI 199 1 ). Whether this phosphorylation at Thrl97 is intra- or intermolecular is not known. The second stable phosphorylation site is Ser338 located close to the carboxy terminus on the strand that wraps around the surface of the small lobe. It is not as rigidly fixed as Thr l 97, at least in the binary and ternary complexes, and is slowly dephosphorylated in vitro during purification (Chiu


440

TAYLOR ET AL

Figure 6

The environment surrounding Thr197. Three residues ligand to the phosphate of Thr(P) 197 and presumably account fur its extreme resistance to hydrolysis by protein phosphatases.

& Tao 1 978; Toner et al 1985). Two additional sites of autophosphorylation, SerlO and Serl39, have been identified in the recombinant protein (W . Yonemoto et ai, manuscript in preparation). The phosphorylation of Ser l O was reported previously i n the mammalian enzyme (Toner et a l 1985). I n the crystal structure of the binary and ternary complex , the amino terminal residues , including Ser l O, are not visible. Ser l 39 lies between helix D and E and is phosphorylated in the crystal structure (D. R. Knighton et aI, unpublished results). THE CATALYTIC SUBUNIT IS A STRUCTURAL FRAMEWORK FOR THE PROTEIN KINASE FAMILY For nearly two decadcs after the elucidation of protein phosphorylation as a mechanism for regulating the activity of glycogen phosphorylase and glyco gen synthase (Fischer & Krebs 1955 ; Friedman & Lamer 1 963) , relatively few protein kinases were known. However, this began to change dramatically when it became clear not only that there were a large number of protein kinases (Hunter 1 987b) , but also that these enzymes were all evolutionarily related (Barker & Dayhoff 1982; Hanks et aI 1 988). Over 200 protein kinases


441

are now known, and the number continues to grow . The general diversity of these enzymes has already been discussed . Here we focus on the similarities that are shared by the entire family of protein kinases and demonstrate why one protein kinase structure can serve as a general framework for the entire family.


Boundaries of the Catalytic Core The boundaries of the catalytic core were defined initially on the basis of the sequence similarities shared by all members of the protein kinase family (Figure 7). Some of the smaller protein kinases also support the assignment of these boundaries and demonstrate that the N-terminal 40 residues and the C-terminal 50 residues in the catalytic subunit are not conserved features in the overall family of protein kinases . The "i-subunit of phosphorylase kinase, for example, begins with what would correspond to residue 25 in the catalytic subunit of cAMP-dependent protein kinase (cAPK) (Reimann et al 1 984) . One of the shortest of the protein kinases is cdc2 (Hindley & Phear 1 984) . It begins with the equivalent of residue 4 1 in the catalytic subunit and ends with the equivalent of residue 30 1 . It should be noted that both of these small protein kinases, the "i-subunit of phosphorylase kinase and cdc2, exist in their active state as part of a multisubunit complex. The closest homologue to cAPK is cGMP-dependent protein kinase. In this case, the cGMP-binding domain and the catalytic domain are part of a contiguous polypeptide chain with the catalytic domain at the carboxy terminal end (Takio et al 1 984b). The cGMP-binding domain immediately precedes the catalytic core region and does not allow for the N-terminal helix found in cAPK (Weber et al 1 989) . In the case of cGPK, the catalytic core must begin with l3-strand 1 . Other kinases, such as src (Martinez et al 1 987; Czemilofsky et al 1980) and casein kinase II (Saxena et al 1987), are shorter at the C-terminus than the catalytic subunit and , hence, the C-terminal tail that wraps around the surface of the catalytic subunit, residues 300 to 350, cannot be a conserved feature of all protein kinases. Based both on the comparative sequences of other kinases and on the structure of the catalytic subunit, the amino-terminal border of the conserved core most likely begins near l3-strand 1 or approximately at residue 40. The second intron/exon boundary lies between Gln35 and Asn36 and is also consistent with this site being the beginning of a conserved structural motif (Chrivia et aI 1 988) . The C-terminal boundary extends at least through residue 280, the last of the invariant residues, and probably extends an additional 20 residues through the I-helix (D. R. Knighton et al 1 992). Many residues important for peptide recognition lie in the core; however , some, such as Glu3 3 1 in the catalytic subunit, lie outside the core. In the case of MLCK, some residues thought to be important for peptide recognition lie

442

TAYLOR ET AL �1

eAJtP

Pltcf} KLeK PBOsy cdc2 EGJ'R

�2

�

PKC �

MLeK PROSy


cdc2

J

-s;:

aC

�

,II;

,.

13J9/LTDrKrLMVLGItGSrGItVMLSBRItG----TDSLYAVItILItKDVV�Q�------DD�e�.va.aVLA�PG / S 1 5 /SD VYIU ••RLGSGItFGQVFRLVEItIt----TGItV'U,GItI'I'It.l..ys.a.-_ ..}· - - -- -ItWU.XI\Da:t$:rJl.CLB ' 1015 IURUP KIILGRGVSSVVRRCIBI

cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes.

The protein kinase C family.

Correction: Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization.

Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization.

Structural and Energetic Characterization of the Ankyrin Repeat Protein Family.

Deciphering the structural basis of eukaryotic protein kinase regulation.

The MARCKS brothers: a family of protein kinase C substrates.

The trk family of tyrosine protein kinase receptors.

Diversity of the protein kinase gene family in rice.

Paralog-Specific Patterns of Structural Disorder and Phosphorylation in the Vertebrate SH3-SH2-Tyrosine Kinase Protein Family.

Viral Evolved Inhibition Mechanism of the RNA Dependent Protein Kinase PKR's Kinase Domain, a Structural Perspective.

A deep learning framework for modeling structural features of RNA-binding protein targets.

Structural basis for the inhibition of host protein ubiquitination by Shigella effector kinase OspG.

Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

Structural Insight into the 14-3-3 Protein-dependent Inhibition of Protein Kinase ASK1 (Apoptosis Signal-regulating kinase 1).

Family-wide Structural Analysis of Human Numb-Associated Protein Kinases.

Family Environment and Childhood Obesity: A New Framework with Structural Equation Modeling.

Protein Kinase D family kinases: roads start to segregate.

A distinctive family of embryonic protein-tyrosine kinase receptors.

Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).

Structural insights into G protein-coupled receptor kinase function.

Structural and Functional Characterization of the VQ Protein Family and VQ Protein Variants from Soybean.

Homeodomain-interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito.

Structural Insights into the Molecular Recognition between Cerebral Cavernous Malformation 2 and Mitogen-Activated Protein Kinase Kinase Kinase 3.