Dynamics of signaling by PKA

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1 Biochimica et Biophysica Acta 1754 (2005) Review Dynamics of signaling by PKA Susan S. Taylor *, Choel Kim, Dominico Vigil, Nina M. Haste, Jie Yang, Jian Wu, Ganesh S. Anand Department of Chemistry and Biochemistry and Department of Pharmacology, Howard Hughes Medical Institute, University of California San Diego, 9500 Gilman Drive, La Jolla, CA , USA Received 25 June 2005; received in revised form 11 August 2005; accepted 12 August 2005 Available online 22 September 2005 Abstract The catalytic and regulatory subunits of camp-dependent protein kinase (PKA) are highly dynamic signaling proteins. In its dissociated state the catalytic subunit opens and closes as it moves through its catalytic cycle. In this subunit, the core that is shared by all members of the protein kinase family is flanked by N- and C-terminal segments. Each are anchored firmly to the core by well-defined motifs and serve to stabilize the core. Protein kinases are not only catalysts, they are also scaffolds. One of their major functions is to bind to other proteins. In addition to its interactions with the N- and C- termini, the catalytic subunit interacts with its inhibitor proteins, PKI and the regulatory subunits. Both bind with subnanomolar affinity. To achieve this tight binding requires docking of a substrate mimetic to the active site cleft as well as a peripheral docking site. The peripheral site used by PKI is distinct from that used by RIa as revealed by a recent structure of a C:RIa complex. Upon binding to the catalytic subunit, the linker region of RIa becomes ordered. In addition, camp-binding domain A undergoes major conformational changes. RIa is a highly malleable protein. Using small angle X-ray scattering, the overall shape of the regulatory subunits and corresponding holoenzymes have been elucidated. These studies reveal striking and surprising isoform differences. D 2005 Published by Elsevier B.V. Keywords: PKA; PKI; Holoenzyme; RIa; RIIa; RIIh; Signaling; Allostery; Dynamics 1. Introduction The protein kinases, which play such a critical role in regulation in eukaryotic cells, constitute a remarkably sophisticated family of enzymes. In terms of numbers, they are one of the largest superfamilies accounting for just under 2% of the human genome [1]. The multiple splice variants compound the complexity even further. Because so many protein kinases are linked to disease, these enzymes have rapidly become major therapeutic targets. While many protein kinase structures are now solved (more than 60 unique members of the protein Abbreviations: camp, cyclic-3v, 5V-adenosine monophosphate; PKA, camp-dependent protein kinase; PKI, heat stable protein kinase A inhibitor; PDK1, phosphoinositide-dependent kinase-1; ATP, adenosine triphosphate; ADP, adenosine diphosphate; D/D domain, dimerization/docking domain; SAXS, small-angle X-ray scattering; AKIP1, A Kinase Interacting Protein 1; H/ DMS, Hydrogen/deuterium exchange coupled with mass spectrometry; PBC, phosphate binding cassette * Corresponding author. Tel.: ; fax: address: staylor@ucsd.edu (S.S. Taylor). kinase family), our understanding of structure and function is still incomplete. This is not only because the kinases are often part of large complexes but also because these proteins are highly dynamic as they toggle between active and inactive conformations and between different locations in the cell. Thus, achieving full understanding of a protein kinase requires many different methods. For high-resolution information, we depend on crystallography and NMR, not only of the conserved core, but also of the full-length proteins with their associated regulatory domains. However, to appreciate the dynamics, additional solution methods are required. Furthermore, while we can glimpse function from isolated domains, and frequently this is all that is accessible by our highresolution methods, ultimately we must achieve a detailed molecular understanding of the full-length proteins and the physiologically relevant complexes that they form. Finally, we must understand how these molecules function in the context of living cells. The challenges and complexities of the protein kinase family are clear in Fig. 1, showing a simple comparison of six /$ - see front matter D 2005 Published by Elsevier B.V. doi: /j.bbapap

2 26 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) Fig. 1. Protein kinase superfamily. In the center is the Human Kinome (Human Kinome image courtesy of Cell Signaling Technology, Inc. ( [1] where kinases that have crystal structures are indicated by a red dot. On the left are six representative kinases where the conserved fold is seen by the color coded subdomains as described by Hanks and Hunter [2]. On the right is the electrostatic surface of each kinase, created using GRASS [51]. The Protein Kinase Resource where each aligned kinase can be profiled is access through [52]. The kinases shown include the catalytic subunit of PKA (PDB 1atp) [5]; cdk2 (PDB 1hck) [53]; c-src (PDB 2src) [54]; InsR (PDB 1ir3) [55]; PhosK (PDB 1phk) [56]; CK1 (PDB 1csn) [57].

3 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) structures representing different branches of the kinome. As seen on the left, all of these enzymes share the same general fold where the conserved subdomains, as defined by Hanks and Hunter [2], are color coded. It is not the fold nor the mechanism of catalysis that distinguish these enzymes where most of the conserved residues cluster around the active site cleft and contribute to catalysis. Instead it is the surfaces that display such remarkable chemical diversity. It is the surface of each kinase that contributes to protein:protein interactions and mediates the assembly and disassembly of dynamic signaling networks. 2. camp-dependent protein kinase The catalytic subunit of camp-dependent protein kinase (PKA) remains as a prototype for this protein superfamily for many reasons. The catalytic (C) and regulatory (R) subunits are relatively small (40 K and K, respectively), and they can be readily expressed in bacteria as active proteins. Furthermore, the catalytic subunit contains only short flanking regions at the N- and C-termini (39 and 50 residues, respectively) in addition to the conserved core and has been crystallized with both substrates and inhibitors [3 6]. Most stages of the catalytic cycle have been captured in a crystal lattice including a putative transition state complex [7 13]. From these structures, we have gained an appreciation of how the cleft opens and closes to grasp the adenine ring at the base of the active site cleft and how the glycine-rich loop and the catalytic loop position the g-phosphate for transfer to a protein substrate. The rules appear to be rather similar for all of these enzymes. Typically, the rate of phosphoryl transfer is very fast with the rate-limiting step being either release of the ADP or some other conformational change that allows the enzyme to commit to another cycle of catalysis [14 16]. Since these enzymes are molecular switches, it is essential to understand how they are inhibited in addition to understanding how they work as catalysts. This is especially true for PKA where the catalytic subunit is assembled as a fully phosphorylated and active enzyme that is then kept sequestered in an inactive state by its association with a regulatory subunit so that the active protein is released only upon binding of camp. Our goals for this review are to describe the inhibited forms of PKA and to show how molecular features of the inhibitor proteins, both PKI and RIa, contribute to their function. Highlighted will be the dynamic features of the inhibitors as they wrap around the remarkably stable catalytic subunit. The structure of the C-subunit bound to a deletion mutant of the RIa subunit has revealed, in particular, the remarkable utility of the surface of the large lobe of the catalytic subunit [17]. Ithas defined this lobe as a stable docking scaffold and also has demonstrated previously unappreciated hot spots that appear to be critical for recognition. By using hydrogen/deuterium (H/ D) exchange, in parallel with mass spectrometry, we also can begin to appreciate the allosteric network that permeates the molecule and enables changes at distal sites to be sensed at the active site cleft and vice versa [18]. Finally, based on small angle X-ray scattering, we shall describe the shapes of the free R-subunits and the corresponding holoenzyme complexes for RIa, RIIa and RIIh demonstrating how this highly conserved family of proteins assumes very different shapes with the differences dictated in large part by the linker regions that join the dimerization/docking (D/D) domain at the N-terminus to the camp-binding domains at the C-terminus [19,20]. Small angle X-ray Scattering (SAXS) studies of the RIa:C complex [21], in addition to of heterodimer complexes of RIa, RIIa, and RIIh [22] reveal major conformational changes in the positioning of the B domain that accompany holoenzyme formation in RIa. This highly dynamic molecular architecture adds a new dimension to our thinking about PKA. 3. General architecture of the catalytic and regulatory subunits The overall architecture of the regulatory and catalytic subunits of PKA are summarized in Figs. 2 and 3. The C- subunit, in addition to its catalytic core (residues ), contains an extension of 39 residues at the N-terminus and 50 residues at the C-terminus. As seen in Fig. 2, both tails interface with the core in ways that contribute to function [8,9,23]. The C-terminal tail is anchored firmly to the large lobe by residues and to the small lobe by the C- terminal hydrophobic cap (residues ). These two motifs are actually highly conserved in all AGC kinases. The highly dynamic, intervening segment, residues , plays a role in the recognition of peptide/protein substrates. The N-terminus, through Trp30, stabilizes the hinge between the small and large lobes and is also anchored to a hydrophobic pocket on the large lobe by the N-terminal myristyl moiety. Here we would like to emphasize the importance of the N- and C-terminal tails for mediating other protein:protein interactions. For example, the C-subunit is likely phosphorylated in vivo by PDK1 by a mechanism that involves anchoring of the C-terminal hydrophobic motif to the hydrophobic pocket on the small lobe of PDK1 [24,25]. The N-terminus is the site of many covalent modifications including myristylation [26], phosphorylation [27] and deamidation [28]. In addition, we recently identified a protein, termed A Kinase Interacting Protein 1 (AKIP1), that binds to the A helix (residues 15 30) and helps to target the protein to the nucleus [29]. The multiple splice variants that are associated with the first exon of the catalytic subunit also suggest that this segment will play an important role in defining localization and/or other protein:protein interactions [30]. We have already demonstrated that this segment behaves as an isoform-specific myristylation switch whereby binding to the RIIh subunit enhances the dynamic properties of the N- terminus and also positions the myristyl moiety to interact with lipid vesicles [31]. The regulatory subunits (Fig. 3) have a well-defined domain structure with a stable dimerization/docking (D/D) domain at the N-terminus that provides a docking site for the A Kinase Anchoring Proteins (AKAPs). The D/D is joined to camp binding domain A by an extended highly disordered linker that

4 28 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) Fig. 2. Organization of the catalytic subunit. In panel A, a space filling model of the core alone (residues ) with ATP in red is in the center. The small lobe (residues ) is in white and the large lobe, in tan. On either side is the full-length protein with the N-terminus shown in gold on the left and the C-terminus shown in gold on the right. Below are ribbon diagrams that highlight the N- and C-termini. Panel B shows the N-terminus (residues 1 39) in red and the AKIP binding surface. In Panel C, the C-terminus (residues ) is highlighted. contains an autoinhibitory sequence. Several putative phosphorylation sites as well as an SH3 binding motif are also found in the linker regions. Like the D/D domain, both camp binding domains are well-folded, stable domains. At the C- terminus of the RIa subunit is a putative PDZ motif. In the absence of C-subunit, the linker region is quite flexible based on fluorescence anisotropy studies using unique sites for Cys labeling [32]. Some of the linker, but not all, becomes ordered upon binding to the C-subunit. Through our SAXS studies, we now recognize that the linker regions contribute in significant and unique ways to the overall architecture of the free R- subunits and of their corresponding holoenzymes Catalytic subunit as a docking scaffold The ability to appreciate the C-subunit as a scaffold for docking to different proteins was highlighted first by the way that the N- and C-terminus fold over and interact with the small and large lobes of the core (Fig. 2). The first 15 residues of the C- terminal extension interact in a stable way with the large lobe while the next 20 residues are quite dynamic and contribute both to peptide binding and catalysis. The C terminal hydrophobic motif is firmly anchored to the small lobe, specifically to the C Helix where it almost certainly contributes to opening and closing of the active site cleft. The structure of a high affinity inhibitor

5 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) Fig. 3. Organization of the regulatory subunits of PKA. On the right is a cartoon of the domain organization of the RIa and RIIh subunits. The sequences of the linker regions are included. On the left is a model of the RIa subunit showing the structured domains and the flexible linker. The D/D domain structure was solved by NMR [44 46] and the cyclic nucleotide binding domains (CBDs) were solved by crystallography [47,48]. The flexibility of the linker was demonstrated by fluorescence anisotropy where the red dots indicate sites where the fluorescent probes are attached [32]. peptide, PKI(5 25), revealed how a peptide docks to the active site cleft and weaves together the two domains in a tight complex that is poised for catalysis [3 5]. It also demonstrated, however, that a second distal or peripheral hydrophobic site on the large lobe was required for high affinity binding [33]. With the RIa:C structure, we see a common mechanism [17].BothRIa and PKI have a pseudoinhibitor site where the P site residue is replaced with Ala. Both require ATP and two Mg ++ ions to form a stable high affinity complex [34]. However, the RIa subunit uses an additional peripheral site to achieve its high affinity binding. In both cases, the catalytic subunit serves as a stable scaffold. The common site and the two distinct peripheral tethering sites used by RIa and PKI are shown in Fig. 4. In the absence of a crystal structure, we identified the interacting surface between the RIa and C-subunits using Fig. 4. Docking surfaces of the catalytic subunit. PKI and RIa share a common inhibitor peptide with PKA substrates. This segment docks to the active site cleft (magenta). In addition, to achieve high affinity binding, each protein requires a second peripheral docking site. The PKI peripheral recognition site (PRF1) (blue) is distinct from the docking surface that is used by RIa (PRF2) (red). (Figure originally published in Kim, C., et al., Science [17].) hydrogen/deuterium exchange coupled with mass spectrometry and a powerful computational docking program [35]. While these studies could not predict major conformational changes in the R-subunit, the docking site of the C-subunit was accurately identified based on the subsequent crystal structure of a deletion mutant of RIa bound to C. Surprisingly, a similar analysis of the C and RIIh subunits, reveals a different docking surface even though both proteins share a conserved domain organization (Law, D., manuscript in preparation). To confirm this important isoform difference, the structure of an RII subunit bound to C needs to be solved. 4. Structure of the RIA(91 244) bound to the C-subunit The catalytic subunit was co-crystallized initially with an inhibitor peptide from PKI, PKI(5 24) [4], and most recently with a deletion mutant of RIa, RIa(91 244), that begins with the inhibitor site in the linker and ends with the C Helix in camp binding domain A [17]. Previous deletion studies showed that this was the smallest fragment of RIa that retained high affinity binding to both C and camp [36]. Although nearly 3000 Å of surface are masked in the complex, the C-subunit remains remarkably stable with the exception of the closing of the active site cleft. Three parts of the C-subunit contribute to recognition of RIa (Fig. 5). In addition to docking of the inhibitor peptide to the active site cleft, there are two additional features of the surface of the large lobe that are critical for this interaction. These are the G Helix and the activation loop Common inhibitor site The occupancy of the active site cleft by the inhibitor peptide (Arg Arg Gly Ala Ile) is identical to PKI(5 24), as was

6 30 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) Fig. 5. C-subunit with inhibitor site. In panel B is the consensus site peptide that docks to the active site cleft. Key elements that interact with the P 3 and P 2 arginines and the P+1 Ile are shown on either side. The shape of the C-subunit is color codes according to Fig. 2.

7 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) predicted (Fig. 5A, B, C). It should be noted how each residue contributes to integration of the small and large lobes. The two basic residues tie together the P+1 loop through Tyr204 and its interaction with Glu230 at the P-2 site, the catalytic loop (through Glu170 as well as the two catalytic residues, Asp166 and Lys168), the linker region (through Glu127), the D Helix through Arg133, the F Helix (through Glu230), and the C- terminal tail (through Tyr330 and its surrounding negatively charged residues). The P+1 loop stabilizes the entire peptide through its interactions with the backbone of the P site residue, through the interactions of Thr201 with Asp166 and Lys168 at the phosphoryl transfer site, and through docking of the P+1 Ile to the hydrophobic P+1 site. Both ATP and the peptide, as well as two Mg ++ ions, are required to achieve a high affinity complex for PKI and RIa [33,34]. In the case of PKI, the inhibitor peptide is tethered to a hydrophobic pocket by an amphipathic helix that precedes the inhibitor segment [4]. In addition, PKI has a P-6 Arg that interacts with Glu203 in the P+1 loop. In the case of the RIa subunit, it is the adjacent surface, C-terminal to the inhibitor peptide, that provides the high affinity peripheral tethering site. It is here where we can appreciate the role of the G Helix and the activation loop The G helix The G Helix (Fig. 6A, C) is a prominent feature of the large lobe that we believe will be important for protein:protein interactions in many other kinases. Hydrogen/deuterium exchange, coupled with mass spectrometry (H/DMS), has demonstrated the remarkable stability of three helices in the large lobe the E Helix, the F Helix, and the H Helix [18]. Even after 30 h, peptides from these regions barely exchange any of their backbone amides. In contrast, the G Helix is much more solvent accessible. Furthermore, we predicted its importance for binding to the C-subunit when we found that peptides from this region were shielded from deuterium exchange in the holoenzyme complex [35]. Tyr247 in the G Helix is surrounded by hydrophobic residues, and this entire surface is masked by the Fig. 6. Motifs in the large lobe that contribute to recognition of RIa. On the left, some of the major helices of the large lobe are shown. The ae, af, and ah helices are very stable and show little exchange of their backbone deuterons even after 30 h [18]. In contrast, the ag Helix is more solvent accessible. The ag Helix is also shielded from deuteron exchange in the presence of R [35]. On the right, the structure is rotated to show the ag Helix and the activation loop. Two key residues that contribute to docking of RIa are Tyr247 in the ag Helix and Trp196 in the activation loop. The C-subunit of PKA (PDB 1atp) [5] is shown in all panels. (Panel C adapted from Kim, C., et al., Science [17].)

8 32 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) Fig. 7. Hydrophobic interface between the RIa and C-subunit. On the left is the hydrophobic surface of the C-subunit, including Tyr247 in the ag Helix in red. The P+1 Ile in RIa nucleates this surface. On the right is the complementary surface in RIa showing Tyr205 at the tip of the PBC in red. In the center is shown the complementarity of the two surface highlighting the specific interaction of Tyr205 in RIa and Tyr247 in C. These figures also show ordering of the linker region (residues ) and the position of Trp196 in the activation loop of C and the extended B/C Helix in RIa. (Figure originally published in Kim, C., et al., Science [17].) binding of the RIa subunit. As seen in Fig. 7, the hydrophobic interface between the C and RIa subunits is nucleated by the P+1 Ile in the inhibitor site of RIa. In the camp-bound conformation of RIa this residue is highly disordered; however, binding to the C-subunit brings it in close proximity to Tyr247 in the G Helix of the catalytic subunit and to Tyr205 in the phosphate binding cassette of the RIa subunit Activation loop The activation loop, specifically Trp196, is the other region that contributes to the high affinity binding of RIa (Fig. 6B, D). While it was known for some time that phosphorylation of Thr197 on the activation loop was essential for optimizing the activity of the catalytic subunit [37], it was not appreciated until recently that phosphorylation of this loop is also an important prerequisite for binding to the RIa subunit. The role of Trp196 for binding to R was first revealed by a genetic screen where Orellana et al. searched for unregulated phenotypes. Replacement of Trp196 with Arg generated a C-subunit that could no longer be inhibited by the R-subunit [38,39]. More recently, we have demonstrated with a kinase dead mutant, C:(Lys72Arg), that phosphorylation of the activation loop is essential for interacting with the RIa subunit. H/D exchange showed, furthermore, that the phosphorylation of Thr197 could be sensed by all of the loops that surround the active site and contribute to catalysis and ATP binding [40]. Thus the entire dynamic behavior of the protein is altered by the addition of a single phosphate Dynamic malleability of the RIa subunit While nearly 3000 Å are masked when the C-subunit combines with RIa(91 244), the catalytic subunit undergoes minimal conformational change other than a tweaking of the G Helix [17]. This is in striking contrast to the RIa subunit. The structure of the complex between the catalytic subunit and RIa(91 244), shown in Fig. 8, highlights for the first time the surprising malleability of camp binding domain-a. Although this is an ancient signaling domain conserved in every genome, this is the first time we have observed the domain both in the presence of camp and in the presence of another protein. The surprising conformational changes that occur upon binding to the catalytic subunit also predict that the position of the B domain relative to the A domain will be significantly different in the holoenzyme compared to the camp bound conformation. While we have yet to solve a crystal structure of a complex that contains both the A and B domains, our recent small angle X-ray scattering results of the RIa and RIIh heterodimers is consistent with a major change Fig. 8. Conformational malleability of RIa subunit (residues ). The camp-bound conformation is shown on the left. In the center is the complex of RIa(91 244) bound to the catalytic subunit. On the right is the conformation of the RIa(91 244) when it is bound to C. The major change in the B/C helix is highlighted in grey.

9 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) in the conformation and positioning of the B domain in RIa [22]. Fig. 8 compares the conformation of camp-bound RIa (left) with the conformation of the RIa subunit when it is bound to camp (right). RIa undergoes significant conformational change when it binds to the catalytic subunit. The structure of RIa(91 244) when it is free of camp closely resembles what is seen in the camp bound conformation of RIa(91 379) (Kim, C., manuscript in preparation). Thus the dramatic reorganization of the RIa subunit is induced by binding of the catalytic subunit. In addition to ordering of the linker region, which in the dimeric state is disordered, there are two main changes. Most striking is the movement of the B/C Helix of the A domain which is kinked in the camp bound conformation. Upon binding to the catalytic subunit, this segment becomes a fully extended single helix that provides a major interacting surface with the catalytic subunit. Fig. 9 shows the different positions of the B/C Helix in the two conformational states. Five arginine residues contribute in unique ways to each of these structures. In the camp bound conformation, Arg209 is an integral part of the phosphate binding cassette (PBC); it is the docking site for the camp phosphate. In addition, Arg241 binds to Glu200 in the phosphate binding cassette. Glu200 anchors the ribose hydroxyl of camp. In this structure Arg230, Arg231, Lys240 and Arg239 are exposed to solvent. In the holoenzyme complex Arg241 is exposed to solvent while Arg230, Arg231 and Lys240 contribute to the R/C interface. Another critical residue whose importance was not previously appreciated was Tyr205 at the tip of the PBC. This Tyr is facing the solvent in the camp bound conformation but is an integral part of the interface in the complex where it hydrogen bonds to Tyr247 in the G Helix of the catalytic subunit and also serves to nucleate the hydrophobic surfaces that come together in the RIa:C complex. 5. Global architecture of the PKA holoenzymes As was seen in Fig. 3, the regulatory subunits of PKA (RIa, RIh, RIIa, and RIIh) share a common domain organization based on extensive sequence similarity. Nevertheless, the isoforms all have unique phenotypes; they are not functionally redundant [41 43]. Furthermore, the overall architecture of each isoform appears to be quite distinct. Each domain also has multiple functions that contribute in unique ways to protein:- protein interactions and to the overall signaling network. Thus, changes in the global organization of each holoenzyme can have major consequences for the assembly of the larger complexes that are built not only by interactions with other proteins but also by interactions with membranes. The two major stable and well-folded domains in the regulatory subunits are the dimerization/docking domain at the N-terminus and the two tandem camp binding domains at the C-terminus. The overall fold of each domain is conserved, although the sequence is clearly conserved in an isoformspecific manner. The four helix bundle topology of the D/D domain provides a stable docking surface for binding to the amphipathic helix motif that is characteristic of AKAPs. This surface provides an isoform-specific surface for high affinity binding of the AKAPs (1 50 nm) [44 46]. The fold of each camp binding domain is also highly conserved in its camp bound conformation; however, the interface between the A and B domains is different for RIa and RIIh [47,48]. These differences in the domain interface provide for a different allosteric network of communication between domain A and B and for communication with the catalytic subunit. As seen in Fig. 9. Nucleation of RIa:C interface. On the left is the camp bound conformation where Arg241 and Arg209 contribute to anchoring of the camp. The structure shown is R p -camp bound RIa (PDB 1ne4) [58]. On the right is the extended B/C helix when it is bound to C. This extended helix docks to the region of the activation loop that includes Trp196. Lys240, Arg230 and Arg231, exposed to solvent in the camp bound conformation, now contribute to the R/C interface. The structure shown is the C-subunit of PKA bound to RIa(91 244) (PDB 1u7e) [17].

10 34 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) the complex of the catalytic subunit with RIa, the camp binding domains are highly malleable and can undergo a major conformational change that is induced upon binding to the catalytic subunit. To characterize the overall shape of the regulatory subunits and the corresponding holoenzymes, we used a combination of crystallography, NMR, and small angle X-ray and neutron scattering (SAXS and SANS). High-resolution structures of the camp binding domains were solved crystallographically [47,48], while the D/D domains were solved by NMR in collaboration with P. Jennings (UCSD) [44 46]. While the high-resolution structures reveal the chemical details of the domains, the highly disorder linker regions make it extremely difficult to solve structures of the full-length proteins or of the corresponding holoenzymes. To overcome this, we used SAXS and SANS which can reveal the shapes of the full-length proteins in solution at low resolution. The SAXS and SANS studies were carried out in collaboration with J. Trewhella and D. Blumenthal at the University of Utah [19,21,49]. In combination with the structures of the domains, we can begin to reconstruct the overall topology of the holoenzymes. A comparison of the dimeric regulatory subunits shows two strikingly different structures. Although the RI and RII subunits are all quite asymmetric, the RIa homodimer is Y shaped and has a maximum diameter of 140 Å [21]. In contrast, the RIIa and RIIh subunits appear to be fully extended, almost dumbbell shaped, with a maximum diameter of nearly 200 Å [19]. This difference between the RI and RII subunits appears to be due mostly to the linker regions which were not thought previously to play a major role. Certainly one would not predict such a major difference bases solely on the sequence comparison. The results provide new roles for the contributions that the linker regions make to the overall conformation. The fact that there are several putative phosphorylation sites as well as an SH3 binding site also suggests that there may be further ways to regulate the shape and conformation of each holoenzyme. The most surprising difference, however, was revealed when the holoenzymes were compared. The RIa holoenzyme was characterized using both SAXS and then SANS where the RIa subunit was deuterated [21]. By adjusting the deuteron content of the solvent one can blank out the R- and C-subunits so that it is possible to observe the shape of the individual R- and C-subunits in the holoenzyme complex. We found that the RIa subunit undergoes significant conformational change when it associates with the catalytic subunit. A more recent comparison of heterodimers with a deletion mutant of RIa, RIa(92 374), confirms this change and show furthermore that the change results from a major change in the position of the B domain [22]. This is consistent with the structure described above where the major change in the conformation of the B/C Helix would require that the B domain be positioned very differently. The SAXS analysis of the heterodimer confirms this prediction and highlights the importance of the dynamic C Helix as a major determinant of conformational change. The two RII holoenzymes were also compared by SAXS. In the RIIa holoenzyme, the RIIa subunits remains fully extended; the catalytic subunits appear to dock onto the camp binding domains with little subsequent change in conformation [50]. In striking contrast, the RIIh subunit compacts into a nearly globular protein upon complex formation with the catalytic subunits [20]. The modeled structure for the RIIh holoenzyme is shown in Fig. 10. Once again, it appears to be the linker region that accounts for this difference. In contrast to Fig. 10. Model of the RIIh holoenzyme based on Small Angle X-ray Scattering. The catalytic subunit (PDB 1syk) [59] is shown as a space filling model with the N- terminal aa helix highlighted. The position of Lys16, where we engineered a fluorescent label is shown as a red dot [31] the B/C helix of RIIh (PDB 1cx4) [48], predicted to be at the R/C interface is shown in red (Law, D., Manuscript in Preparation). The N terminal myrisyl moiety is indicated as a yellow oval. The position of an AKAP docking helix is shown as a red sphere. The regulatory subunits are shown as turquoise ribbons.

11 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) the poorly conserved linker in RIIa, the linker of RIIh is highly conserved (Fig. 3). In the model it is predicted that both the D/ D domain and the camp binding domains interact in part with the linker region in the holoenzyme complex. The modeled structure shows, furthermore, a striking disposition of the catalytic subunits relative to a potential membrane surface. The N-terminus of the catalytic subunit with its myristylation motif was known to become quite mobile upon formation of a holoenzyme complex with RIIh although in the RIa holoenzyme the N-terminus is even more ordered than in the free C- subunit where the myristyl groups folds into a hydrophobic pocket [31]. Furthermore, unlike the free C-subunit and the RIa holoenzyme, the RIIh holoenzyme associates readily with membranes. The model shows how the positioning of the myristyl moieties in the RIIh holoenzyme could provide for a bivalent contact with membranes. Further, anchoring to membranes can be achieved by binding to the AKAPs, and in the model the AKAP binding surface is on the same surface as the myristylated A Helix. One can also appreciate in this model how the B/C Helix that links the A and B domains in RIIh can bind to the surface of the catalytic subunit in a manner that does not require a conformational change in the RIIh subunit. This is consistent with H/D exchange and docking studies of the RIIh holoenzyme where the RIIh appears to dock to a sight that overlaps with PKI and is quite different from RIa (Law, D., manuscript in preparation). Of course, further studies are needed, including high-resolution structures of larger complexes, to fully appreciate the allosteric mechanisms and chemical pathways that weave these proteins together. The SAXS studies, however, allow us to recognize for the first time that these molecular assemblies are very different for the PKA isoforms. 6. Conclusions and perspectives PKA continues to serve as a prototype for understanding the structure, function and dynamics of this important enzyme family. While previous work has defined the relevant conformational states associated with catalysis, the recent structure of a catalytic subunit bound to a deletion mutant of RIa reveals the extraordinary conformational malleability of the regulatory subunit. Solution studies using small angle neutron scattering add further to our understanding of the dynamic behavior of the regulatory subunits. Surprisingly, and in spite of considerable sequence similarity, the PKA isoforms show striking differences. The challenge for the future will be to characterize large complexes using a combination of crystallography for highresolution together with solution methods such as NMR, hydrogen/deuterium exchange coupled with mass spectrometry, and small angle X-ray and neutron scattering. Only in this way will we be able to appreciate the kinase not only as a catalyst but also as a multivalent scaffold for docking to many other proteins. Acknowledgements We gratefully acknowledge the funding for this work from NIH (GM34921 and GM19301) to S.S.T., from GM34921 to D.V., and from T32-DK07233 to C.K. We acknowledge Cell Signaling Technology, Inc. ( for use of the Human Kinome image (Fig. 1). References [1] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) [2] S.K. Hanks, T. Hunter, Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification, FASEB J. 9 (1995) [3] D.R. Knighton, J. Zheng, L.F. Ten Eyck, V.A. Ashford, N.-h. Xuong, S.S. Taylor, J.M. Sowadski, Crystal structure of the catalytic subunit of campdependent protein kinase, Science 253 (1991) [4] D.R. Knighton, J. Zheng, L.F. Ten Eyck, N.H. Xuong, S.S. Taylor, J.M. Sowadski, Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase, Science 253 (1991) [5] J. Zheng, D.R. Knighton, L.F. Ten Eyck, R. Karlsson, N.-h. Xuong, S.S. Taylor, J.M. Sowadski, Crystal structure of the catalytic subunit of campdependent protein kinase complexed with MgATP and peptide inhibitor, Biochemistry 32 (1993) [6] N. Narayana, T.C. Diller, K. Koide, M.E. Bunnage, K.C. Nicolaou, L.L. Brunton, N.H. Xuong, L.F. Ten Eyck, S.S. Taylor, Crystal structure of the potent natural product inhibitor balanol in complex with the catalytic subunit of camp-dependent protein kinase, Biochemistry 38 (1999) [7] J. Zheng, D.R. Knighton, N.H. Xuong, S.S. Taylor, J.M. Sowadski, L.F. Ten Eyck, Crystal structures of the myristylated catalytic subunit of camp-dependent protein kinase reveal open and closed conformations, Protein Sci. 2 (1993) [8] N. Narayana, S. Cox, S. Shaltiel, S.S. Taylor, N.H. Xuong, Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of camp-dependent protein kinase complexed with the peptide inhibitor PKI(5 24) and adenosine, Biochemistry 36 (1997) [9] N. Narayana, S. Cox, N.-h. Xuong, L.F. Ten Eyck, S.S. Taylor, A binary complex of the catalytic subunit of camp-dependent protein kinase and adenosine further defines conformational flexibility, Structure 5 (1997) [10] Madhusudan, P. Akamine, N.H. Xuong, S.S. Taylor, Crystal structure of a transition state mimic of the catalytic subunit of camp-dependent protein kinase, Nat. Struct. Biol. 9 (2002) [11] P. Akamine, Madhusudan, J. Wu, N.H. Xuong, L.F. Ten Eyck, S.S. Taylor, Dynamic features of camp-dependent protein kinase revealed by apoenzyme crystal structure, J. Mol. Biol. 327 (2003) [12] D.A. Johnson, P. Akamine, E. Radzio-Andzelm, Madhusudan, S.S. Taylor, Dynamics of camp-dependent protein kinase, Chem. Rev. 101 (2001) [13] F. Li, M. Gangal, C. Juliano, E. Gorfain, S.S. Taylor, D.A. Johnson, Evidence for an internal entropy contribution to phosphoryl transfer: a study of domain closure, backbone flexibility, and the catalytic cycle of camp-dependent protein kinase, J. Mol. Biol. 315 (2002) [14] J.A. Adams, Kinetic and catalytic mechanisms of protein kinases, Chem. Rev. 101 (2001) [15] J.A. Adams, S.S. Taylor, Energetic limits of phosphotransfer in the catalytic subunit of camp-dependent protein kinase as measured by viscosity experiments, Biochemistry 31 (1992) [16] J. Lew, S.S. Taylor, J.A. Adams, Identification of a partially ratedetermining step in the catalytic mechanism of camp-dependent protein kinase: a transient kinetic study using stopped-flow fluorescence spectroscopy, Biochemistry 36 (1997) [17] C. Kim, N.H. Xuong, S.S. Taylor, Crystal structure of a complex between the catalytic and regulatory (RIalpha) subunits of PKA, Science 307 (2005) [18] J. Yang, S.M. Garrod, M.S. Deal, G.S. Anand, V.L. Woods Jr., S. Taylor,

12 36 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) Allosteric network of camp-dependent protein kinase revealed by mutation of Tyr204 in the P+1 loop, J. Mol. Biol. 346 (2005) [19] D. Vigil, D.K. Blumenthal, W.T. Heller, S. Brown, J.M. Canaves, S.S. Taylor, J. Trewhella, Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions, J. Mol. Biol. 337 (2004) [20] D. Vigil, D.K. Blumenthal, S.S. Taylor, J. Trewhella, Major Differences in Global Architectures of Type II PKA Isoforms: implications for signaling specificity, J. Biol. Chem. (submitted for publication). [21] W.T. Heller, D. Vigil, S. Brown, D.K. Blumenthal, S.S. Taylor, J. Trewhella, C subunits binding to the protein kinase A RI alpha dimer induce a large conformational change, J. Biol. Chem. 279 (2004) [22] D. Vigil, D.K. Blumenthal, S.S. Taylor, J. Trewhella, The Conformationally Dynamic C Helix of the RIalpha-Subunit of Protein Kinase A Mediates Isoform Specific Domain Reorganization Upon C Subunit Binding, J. Biol. Chem. (in press). [23] S. Shaltiel, S. Cox, S.S. Taylor, Conserved water molecules contribute to the extensive network of interactions at the active site of protein kinase A, Proc. Nat. Acad. Sci. U. S. A. 95 (1998) [24] X. Cheng, Y. Ma, M. Moore, B.A. Hemmings, S.S. Taylor, Phosphorylation and activation of camp-dependent protein kinase by phosphoinositide-dependent protein kinase, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) [25] M.J. Moore, J.R. Kanter, K.C. Jones, S.S. Taylor, Phosphorylation of the catalytic subunit of protein kinase A. Autophosphorylation versus phosphorylation by phosphoinositide-dependent kinase-1, J. Biol. Chem. 277 (2002) [26] S.A. Carr, K. Biemann, S. Shoji, D.C. Parmelee, K. Titani, n-tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) [27] J. Toner-Webb, S.M. van Patten, D.A. Walsh, S.S. Taylor, Autophosphorylation of the catalytic subunit of camp-dependent protein kinase, J. Biol. Chem. 267 (1992) [28] V. Kinzel, N. Konig, R. Pipkorn, D. Bossemeyer, W.D. Lehmann, The amino terminus of PKA catalytic subunit A site for introduction of postranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes, Protein Sci. 11 (2000) [29] M. Sastri, D.M. Barraclough, P.T. Carmichael, S.S. Taylor, A-kinaseinteracting protein localizes protein kinase A in the nucleus, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) [30] S. Orstavik, N. Reinton, E. Frengen, B.T. Langeland, T. Jahnsen, B.S. Skalhegg, Identification of novel splice variants of the human catalytic subunit Cbeta of camp-dependent protein kinase, Eur. J. Biochem. 268 (2001) [31] M. Gangal, T. Clifford, J. Deich, X. Cheng, S.S. Taylor, D.A. Johnson, Mobilization of the A-kinase N-myristate through an isoform-specific intermolecular switch, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) [32] F. Li, M. Gangal, J.M. Jones, J. Deich, K.E. Lovett, S.S. Taylor, D.A. Johnson, Consequences of camp and catalytic-subunit binding on the flexibility of the A-kinase regulatory subunit, Biochemistry 39 (2000) [33] D.A. Walsh, K.L. Angelos, S.M. Van Patten, D.B. Glass, L.P. Garetto, in: B.E. Kemp (Ed.), Peptides and Protein Phosphorylation, CRC Press, Inc., Boca Raton, 1990, pp [34] F.W. Herberg, M.L. Doyle, S. Cox, S.S. Taylor, Dissenction of the nucleotide and metal-phosphate binding sites in camp-dependent protein kinase, Biochemistry 38 (1999) [35] G.S. Anand, D. Law, J.G. Mandell, A.N. Snead, I. Tsigelny, S.S. Taylor, L.F. Ten Eyck, E.A. Komives, Identification of the protein kinase A regulatory RIalpha-catalytic subunit interface by amide H/2H exchange and protein docking, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) [36] L.J. Huang, S.S. Taylor, Dissecting camp binding domain A in the RIalpha subunit of camp-dependent protein kinase. Distinct subsites for recognition of camp and the catalytic subunit, J. Biol. Chem. 273 (1998) [37] J.A. Adams, M.L. McGlone, R. Gibson, S.S. Taylor, Phosphorylation modulates catalytic function and regulation in the camp-dependent protein kinase, Biochemistry 34 (1995) [38] S.A. Orellana, P.S. Amieux, X. Zhao, G.S. McKnight, Mutations in the catalytic subunit of the camp-dependent protein kinase interfere with holoenzyme formation without disrupting inhibition by protein kinase inhibitor, J. Biol. Chem. 268 (1993) [39] S.A. Orellana, G.S. McKnight, Mutations in the catalytic subunit of camp-dependent protein kinase result in unregulated biological activity, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) [40] G. Iyer, S. Garrod, V.L. Woods Jr., S.S. Taylor, Catalytic Independent Functions of a Protein Kianse as Revealed by a Kinase-dead Mutant: study of the Lys72His Mutant of camp-dependent kinase, J. Biol. Chem. (in press). [41] D.E. Cummings, E.P. Brandon, J.V. Planas, K. Motamed, R.L. Idzerda, G.S. McKnight, Genetically lean mice result from targeted disruption of the RII beta subunit of protein kinase A, Nature 382 (1996) [42] P.S. Amieux, D.E. Cummings, K. Motamed, E.P. Brandon, L.A. Wailes, K. Le, R.L. Idzerda, G.S. McKnight, Compensatory regulation of RIalpha protein levels in protein kinase A mutant mice, J. Biol. Chem. 272 (1997) [43] E.P. Brandon, R.L. Idzerda, G.S. McKnight, PKA isoforms, neural pathways, and behaviour: making the connection, Curr. Opin. Neurobiol. 7 (1997) [44] M.G. Newlon, M. Roy, D. Morikis, D.W. Carr, R. Westphal, J.D. Scott, P.A. Jennings, A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes, EMBO J. 20 (2001) [45] M.G. Newlon, M. Roy, D. Morikis, Z.E. Hausken, V. Coghlan, J.D. Scott, P.A. Jennings, The molecular basis for protein kinase A anchoring revealed by solution NMR, Nat. Struct. Biol. 6 (1999) [46] P. Banky, M. Roy, M.G. Newlon, D. Morikis, N.M. Haste, S.S. Taylor, P.A. Jennings, Related protein protein interaction modules present drastically different surface topographies despite a conserved helical platform, J. Mol. Biol. 330 (2003) [47] Y. Su, W.R.G. Dostmann, F.W. Herberg, K. Durick, N.-h. Xuong, L.F. Ten Eyck, S.S. Taylor, K.I. Varughese, Regulatory (RIa) subunit of protein kinase A: structure of deletion mutant with camp binding domains, Science 269 (1995) [48] T.C. Diller, Madhusudan, N.H. Xuong, S.S. Taylor, Molecular basis for regulatory subunit diversity in camp-dependent protein kinase: crystal structure of the type II beta regulatory subunit, Structure (Camb) 9 (2001) [49] D. Vigil, D.K. Blumenthal, S. Brown, S.S. Taylor, J. Trewhella, Differential effects of substrate on type I and type II PKA holoenzyme dissociation, Biochemistry 43 (2004) [50] J. Zhao, E. Hoye, S. Boylan, D.A. Walsh, J. Trewhella, Quaternary structures of a catalytic subunit-regulatory subunit dimeric complex and the holoenzyme of the camp-dependent protein kinase by neutron contrast variation, J. Biol. Chem. 273 (1998) [51] M. Nayal, B.C. Hitz, B. Honig, GRASS: a server for the graphical representation and analysis of structures, Protein Sci. 8 (1999) [52] The Protein Kinase Resource: an integrated environment for phosphorylation research, PROTEINS: structure, Function, and Bioinformatics (in press). [53] U. Schulze-Gahmen, H.L. De Bondt, S.H. Kim, High-resolution crystal structures of human cyclin-dependent kinase 2 with and without ATP: bound waters and natural ligand as guides for inhibitor design, J. Med. Chem. 39 (1996) [54] W. Xu, A. Doshi, M. Lei, M.J. Eck, S.C. Harrison, Crystal structures of c- Src reveal features of its autoinhibitory mechanism, Mol. Cell 3 (1999) [55] S.R. Hubbard, Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog, EMBO J. 16 (1997) [56] D.J. Owen, M.E. Noble, E.F. Garman, A.C. Papageorgiou, L.N. Johnson, Two structures of the catalytic domain of phosphorylase kinase: an active

13 S.S. Taylor et al. / Biochimica et Biophysica Acta 1754 (2005) protein kinase complexed with substrate analogue and product, Structure 3 (1995) [57] R.M. Xu, G. Carmel, R.M. Sweet, J. Kuret, X. Cheng, Crystal structure of casein kinase-1, a phosphate-directed protein kinase, EMBO J. 14 (1995) [58] J. Wu, J.M. Jones, X. Nguyen-Huu, L.F. Ten Eyck, S.S. Taylor, Crystal structures of RIalpha subunit of cyclic adenosine 5V-monophosphate (camp)-dependent protein kinase complexed with (Rp)-adenosine 3V,5Vcyclic monophosphothioate and (Sp)-adenosine 3V,5V-cyclic monophosphothioate, the phosphothioate analogues of camp, Biochemistry 43 (2004) [59] J. Wu, J. Yang, Madhusudan, N.H. Xuong, L.F. Ten Eyck, S.S. Taylor, Crystal structure of E230Q mutant of camp-dependent protein kinase reveals unexpected apoenzyme conformation, Protein Science (in press).