A Review of camp-dependent Protein Kinase A Catalytic Subunit Structure, Function and Regulation

Transcription

1 A Review of camp-dependent Protein Kinase A Catalytic Subunit Structure, Function and Regulation Kaitlyn McLeod* Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA Abstract Word Count: 172 Main Body Word Count: 2137

2 Abstract Protein Kinase A (PKA) catalyzes phosphoryl group transfer by orienting a protein substrate and ATP molecule in the active site cleft of the catalytic subunit, making it an integral part of many cell signaling pathways. Homology studies have identified PKA as a template for all kinases because it represents the conserved core, although specifically, it is part of the AGC kinase group in the human kinome. These conservations focus on residues in the active site and an internal architecture called the C-spine and R-spine. X-ray crystallography studies have been used to investigate the details of the catalytic mechanism Magnesium ions in the active site have emerged as important players in the positioning of the gamma-phosphate of ATP for transfer. PKA is regulated by both phosphorylation and myristylation; these regulation strategies contribute to the molecular switch functioning of PKA rather than efficacious catalyst. Furthermore, the important role of phosphorous in biology as a regulator for other proteins and the integral role PKA has in signaling pathways makes it a target of drug design.

3 Introduction Phosphorylation plays a regulatory role in many biological processes. Phosphate is negatively charged, and thus, by transferring the gammaphosphate from ATP to the hydroxyl groups of serine, threonine, and tyrosine by kinases, conformational adjustments cause enzyme to be active or inactive. Protein Kinase A (PKA), for instance, only achieves an active catalytic state after being phosphorylated at Thr197 and Ser338. Phosphorylation can also indicate a binding site or alter the conformation of a domain to limit interactions of a protein with a ligand. Despite the ability of camp-dependent protein kinase A to catalyze the transfer of phosphate groups, it mainly acts as a molecular switch by regulating the activation of other enzymes (Taylor et al., 2013). PKA is tetrameric with two regulatory units and two catalytic subunits. PKA is downstream of the signal cascade initiated when a hormone binds to a G- Protein Coupled Receptor (GPCR). Because GPCRs are very common transmembrane proteins, PKA is not specific to a pathway. However, there is some evidence that binding of regulatory units near certain GPCRs can lead to specialization. Upon binding a hormone, GPCRs undergo a conformational change that release guanine diphospate (GDP) in the s-alpha subunit of the G- protein attached to the receptor. As guanine triphosphate (GTP) replaces GDP, the activated G s-alpha subunit separates and binds to adenylyl cylclase, an enzyme that catalyzes the cyclization of ATP to cyclic adenosine monophosphate (camp). camp is a secondary messenger that binds regulatory units, releasing

4 PKAc catalytic PKA (Nelson and Cox, 2013). Regulatory subunits bind in the substrate-binding cleft (Figure 1), inhibiting catalytic activity (see Figure 2). Key structural conservations between PKA and related kinases contribute to three primary functions of catalysis: binding and orienting ATP and substrate, transferring the gamma-phosphate of ATP to the substrate. Among these are twelve conserved subdomains (Hanks et al., 1988). Domains one through four make up the N-lobe of the catalytic subunit, also called the small lobe. Named for its proximity to the N-terminus, the N-lobe has one alpha helix and many betasheets and turns; it is malleable and binds ATP. The C-lobe, named for its proximity to the carboxyl-terminus, is mostly of alpha helices; thus, it is much less flexible than the N-lobe (see Figure 4). The interface between the N- and C-lobe positions the substrate and ATP molecule for group transfer. This review of the PKAc attempts to connect the conservation of key residues with specific structural and catalytic functions.

5 Main Body Conservation of Key Amino Acid Residues Many comparison studies have been done on the kinase family to understand the phylogeny of the kinases. camp-dependent PKA is part of one of the largest families of homologous kinases known; this superfamily is divided into two categories: serine/threonine kinases and tyrosine kinase (Hanks et. al 1995). Hanks et al (1988) noted 12 conserved subdomains in a study comparing 65 sequences of both serine/threonine and tyrosine kinases. The conserved residues were labeled according to camp-dependent protein kinase alpha form: Gly52 (subdomain I), Lys72 (subdomain II), Glu91, Asp166 (subdomain VI), Asn171 (subdomain VI), Asp184 (subdomain VII), Gly186 (subdomain VII), Glu208 (subdomain VIII), Arg280, Gly50 (subdomain I), Val57 (subdomain I), Phe185 (subdomain VII), Asp220 (subdomain IX), and Gly225 (subdomain IX) (Hanks, 1988) (Figure 6). Many of these residues are relatively close the ATP binding site and the phosphate transfer site, so they may play a role in catalysis. Subdomain I has the glycine-rich loop that lies above the ATP binding site and has a key residue, Ser53, that interacts with the gamma-phosphate of the ATP molecule, the phosphate that transferred to the substrate. While Asp166, Asn171, Asp184, and Lys72 are featured in the active site and coordinate ATP binding through electrostatic attractions with the serine residue of the substrate and the negatively charged gamma-phosphate. Although sequence comparisons have lead Hank et al. (1988) to identify many functionally important residues, x-ray crystallography structures give insight

6 that may have otherwise been impossible to deduce. Non-consecutive amino acids have been conserved that constitute an internal structure to the kinase called the C-spine (Masterson et al 2010). The C-spine has amino acids from both the N-lobe and the C-lobe that are mostly hydrophobic in nature; one disulfide bond stabilizes the C-spine (see Figure 3), which is anchored to the F- helix that runs through the center of the C-lobe. ATP binding fulfills the C-spine; this explains why Masterson et al (2010) found that PKA was activated upon ATP binding and that Cheng et al. (1986) saw the rigidity of the enzyme increases upon nucleotide binding. The R-spine is known as the regulation spine. It is incomplete in many inactive kinases, and therefore, is thought to be one of the key elements contributing to an active PKA (Taylor et al., 2013) Figure 3 shows the intact R- spine, featuring a vertical alignment. The discovery of the R-spine is attributed to molecular modeling because these residues are again not adjacent in the primary sequence. Furthermore, PKA has the conserved fold that is exhibited in the entire family of kinases. Like the C-spine, both the N- and C-lobes contribute amino acids to the R-spine, and it is connected to the foundational F-helix. This connection is through a hydrogen bond between either a tyrosine or histidine residue at the lower part of the R-spine and Asp220 (Taylor et al., 2013). Note that Asp220 is among the conserved amino acids found in sequence comparison by Hanks et al. (1988). Furthermore, the alignment of the R-spine brings the key residues involved in catalysis and group transfer into close proximity.

7 Regulation of PKA Recent structural studies of PKA have been focused on the regulation of PKA by phosphorylation. Two phosphorylation sites, Ser338 and Thr197, have been identified as essential to enzymatic activity of PKA. Ser338 is located near the C-terminus and is phosphorylated co-translationally; it is not conserved in all PKA structures; thus, Montenegro et al. (2012) suggests that it contributes to substrate selection. The absence of a phosphorylated Ser338 during translation is deleterious to the formation of an active kinase. Although the end of the N-lobe sequence does not have a sequence complementary to the active site, it relies on the phosphorylation of Ser338 to reorder the N-lobe so that the Ser338 is towards the outside of the protein (Taylor et al., 2013). See Figure 4. Montenegro et al. (2012) confirms this stating that replacing Ser338 with alanine destabilized the enzyme. Therefore, Ser338 plays an important role in correct protein folding. Phosphorylation of Thr197 creates a hydrogen bond with Arg165; the proximity of Arg165 to the Asp 166, allows phosphorylation of Thr197 to directly affect the arrangement of the active site (Montenegro et al., 2012). In fact, the same study found that enzymes lacking a phosphorylated Thr197 could not obtain an active catalytic site (Montenegro et al., 2012). Two additional sites of phosphorylation were found by Yonemoto et al. (1993) studying PKA expressed in E Coli: Ser10 and Ser139. Ser10 has been implicated in further studies of N-myristylation. N-myristylation is the addition of a fatty acid chain, myristic acid, to the N-terminal PKAc, which stabilizes the enzyme through hydrogen bonding and has a role in directing PKA to the

8 membranes (Bastidas et al., 2012). When purified from tissues, autophosphorylation of Ser10 was not seen; however, PKA purified from recombinant expression systems phosphorylated Ser10 was observed (Bastidas et al., 2012). Yonemoto et al. (1993) and Bastidas et al. (2012) also noted that phosphorylation of Ser10 and N-myristylation has not been observed simultaneously. This was supported by the findings that N-myristylation orders the N-terminus, while NMR studies showed disordering of the N-terminus by phosphorylated Ser10. N-myristylation may also affect substrate specificity by stabilizing the A- helix that runs between the two lobes of the catalytic subunit (Bastidas et al., 2012). Comparing the apo-enzyme and ternary complex (ATP and peptide bound) of myristylated and non-myristylated subunits showed that myristylation limits the major conformational changes of the enzyme (Bastidas et al., 2013). This is important for substrate binding and contributes to stabilizing a conformation suitable for catalysis. Role of Magnesium in the Active Site Prior to solved structures for PKA, the presence of magnesium in the active site was determined by kinetic studies. Similar dissociation constants of ATP and Mg-ATP complex suggested the first magnesium ion entered the ATPbinding site as an Mg-ATP complex (Armstrong et al. 1979). Adams (2001) noted that this was consistent with x-ray crystallography experiments: at low concentrations of Mg, one ion was bound; at concentrations above physiological conditions, two bound magnesium ions were bound in the active site (Figure 5).

9 Kovalesky et al. (2012) showed that at low magnesium ion concentration, one bound magnesium ion increased ATP mobility. The gamma-phosphate is held in place by hydrogen bonds with Ser53, Lys72, and Asp166 (Figure 5), but the magnesium ions add to the specific positioning of the gamma-phosphate. Kovalesky et al. (2012) further notes that inhibitor binding (IP 20, see below) can occur without the presence of magnesium ions. Thus, the main role of magnesium is stabilizing ATP, not binding inhibitor or substrate. It follows that the affinity of ATP for PKAc increases with increasing metal concentration (Kovalesky et al, 2012). Further studies have investigated whether magnesium is necessary for catalytic activity or if other divalent metals, Ca 2+, Sr 2+, and Ba 2+, can also support enzymatic activity (Gerlits et al., 2013). The atomic radii Ca 2+, Sr 2+, and Ba 2+ are larger than that of Mg 2+, which creates a larger distance between substrate and gamma-phosphate in the active site. Ca 2+ and Sr 2+ were capable of binding ATP, but did not successfully allow for phosphotransfer except at concentrations of great excess to the enzyme (Gerlits et al., 2013). Ba 2+ was not able to promote ATP binding, probably due to size (Gerlits et al., 2013). Although any divalent metal could likely support catalysis, the architecture and space within the active site is limiting. Catalytic Mechanism Pre-steady state kinetic experiments by Zhou and Adams (1997) showed that catalysis by PKAc was not affected by the ph range of 6-9; overall, this data refutes a previous understanding that Asp166, oriented between the gamma-

10 phosphate and substrate hydroxyl, was a general-base catalyst. Due to strict conservation of Asp166, many studies have focused on determining its role. Asp166 hydrogen bonds with the serine of the substrate, orienting the nucleophilic oxygen towards phosphate (Adams, 2001). Through an Sn2 mechanism, the phosphoryl group is transferred and a divalent magnesium ion stabilizes the negative charge (Gerlits et. al, 2013). This mechanism is also consistent with the finding that at high magnesium concentrations, the ratelimiting step is the release of ADP because more electrostatic interactions are made between ADP and magnesium (Gerlits et al. 2013). Further computational studies also have suggest that Asp166 helps shape the active site, while Lys168 has a more direct affect on catalysis (Montenegro et al. 2013). Despite having some clues to the mechanistic details of PKAc, there is still debate. Inhibition and Substrate Recognition Sequences for Possible Drug Targets Because enzymes play key roles in signal cascades, they have been the target of drug design. Protein kinase mutations have been linked to many diseases and are therefore popular drug targets (Einstein and Eldar-Finkelman, 2009). Recent studies have been focused on the inhibition of PKA. Although protein kinase A generally works on proteins, not peptides, peptides that mimic the recognition site of the protein have been generated as competitive inhibitors (Eldar-Finkelman and Einstein, 2009). Furthermore, the endogenous inhibitor of protein kinase A, protein kinase inhibitor (PKI), has been used as a template for designing potential competitive inhibitors.

11 PKA is a basophilic kinase and prefers positively-charged substrates (Eldar-Finkelman and Einstein, 2009). Considering the key active site amino acid residues, Asp166 and Asn171, this is logical. Through independent studies, two primary peptides have been tested: IP 20 and Kemptide. IP 20 was discovered by protease digestion of the natural inhibitor PKI; one of the fragments retained 30% activity, and is referred to as IP 20 (Cheng et al. 1986). The sequence for IP 20 is TTYADFIASGRRNAIHD (Cheng et al., 1986). It has been suggested that the arginine-arginine portion of IP 20 is key in its inhibition. Kemptide s sequence, LRRASLG, also has the duplicated arginine, but acts more as a substrate than an inhibitor, having a K m value of 5uM (Cheng et al., 1986). This substrate activity is due to the presence of serine, a phosphoacceptor. IP 20 has the duplicate arginine sequence that presumably encourages binding to the active site cleft, but lacks a serine or threonine to accept a group transfer. The recognition sequence for the peptide in the catalytic cleft is RRAS; further studies using NMR and X-ray crystallography have shown that the sequence is flexible in solution, but gains rigidity when bound, aiding binding specificity (Cheng et al., 1986). Discussion A variety of techniques were applied to make the discoveries about PKA discussed in this review. Finding ways to observe the behavior of PKA without altering the structure of the protein excessively is a challenge. For instance, X- ray crystallography studies can only provide a snap shot of the enzyme, but cannot show the catalytic activity. Furthermore, it is difficult to isolate desired conformations (open, closed, ternary versus binary complexes) with x-ray

12 crystallography. The specific role of individual residues was studied with sitedirected mutagenesis studies and computational studies (molecular dynamics simulations). These computational studies were integrated with structural studies to attempt to provide mechanistic details. However, in general, the mechanistic details of PKA continue to be debated. PKA represents the conserved core of the kinase superfamily. Many amino acids have been conserved in all kinase sequences compared (Hanks, 1988). These residues tend to be centered around the catalytic core. The conserved kinase core may suggest similar mechanism between kinases; however, these kinase active sites lack the catalytic triad common to many enzymes. Furthermore, research that is trying to solidify the catalytic mechanism of PKA is challenging the general understanding of Michaelis-Menten kinetics (Taylor et al, 2013). Regulation of PKA has been a focus in recent years. The catalytic domain has a number of phosphorylation sites, including Ser10, Ser138, Ser338, and Thr197. Perhaps most importantly, Thr197 is absolutely essential for enzyme activity, while the serine phosphorylation sites seem to play more of a stabilization role (Bastidas et al., 2012; Montenegro et al., 2012). Recent studies are looking into the effect of N-myristylation on enzyme stability and substratebinding specificity (Bastidas et al., 2013; Bastidas et al., 2012). Ultimately, Bastidas et al. (2013) investigation of the effects of N-myristylation suggests that A-helix running along the length of the may have more of an affect on the active site than previously thought.

13 The role of magnesium ions in the catalytic mechanism can be summarized as follows: magnesium is necessary for ATP binding, but not substrate binding (Gerlits et al., 2013). Thus, magnesium is necessary for enzymatic activity; however, all alkaline metals in excess can support phosphotransfer (Gerlits et. al, 2013). Due to the biological relevance of cell signaling and signal cascades, PKA has been implicated as a drug target. The recognition sequence of substrates to PKA has been established and the endogenous inhibitor has been studied (Cheng et al. 1986); this allows for effective design of competitive inhibitors. Drug design can have an important affect on diseases related to kinase mutations. However, the high level of conservation between kinases may prove challenging in designing selective drugs. Acknowledgements Gratitude is extended to Dr. Chad Park and Dan Lajoie for their technical assistance Pymol and guidance in constructing literature reviews.

14 References Armstrong, R. N.; Kondo, H.; Granot, J.; Kaiser, E. T.; Mildvan, A. S. Biochemistry 1979, 18, Bastidas, A. C., Deal, M. S., Steichen, J. M., Keshwani, M. M., Guo, Y., Taylor, S. S. (2012) Role of N-terminal Myristylation in the Structure and Regulation of camp-dependent Protein Kinase. J. Mol. Biol. 422, Bastidas, A. C., Pierce, L. C., Walker, R. C., Johnson, D. A., Taylor, S. S. (2013) Influence of N-Myristylation and Ligand Binding on the Flexibility of the Catalytic Subunit of Protein Kinase A. Biochemistry 52 (37): Cheng, H., Kemp, B., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., Walsh, D. A. (1986) A Potent Synthetic Peptide Inhibitor of the camp-dependent Protein Kinase. J. Biol. Chem. 261, Eldar-Finkelman, H., Einstein, M. (2009) Peptide Inhibitors Targeting Protein Kinases. Current Pharmaceutical Design 15, 1-7. Gerlits, O., Waltman, M.J., Taylor, S., Langran, P., Kovalevsky, A. (2013) Insights into the Phosphoryl Transfer Catalyzed by camp-dependent Protein Kinase: An X-ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20. Biochemistry 52, Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1988; 241(4861): Hanks SK, Hunter T. Protein kinases. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classi- fication. Faseb J 1995; 9(8): Kovalesky, A.Y., Johnson, H., Hanson, L., Waltman, M., Fisher, S., Taylor, S., Langan, P. (2012) Low- and room-temperature X-ray structures of protein kinase A ternary complexes shed new light on its activity. Acta Cryst. D68, Masterson, L. R., Cheng, C., Yu, T., Tonelli M., Kornev, A., Taylor, S., Veglia, G. (2010) Dynamics connect substrate recognition to protein kinase A. Nature Chemical Biology 6, DOI: Montenegro, M. Masgrau, L., Gonzalez-Lafont, A., Lluch, J., Garcia-Vioca, M. (2012) Influence of enzyme phosphorylation state and the substrate on PKA enzyme dynamics. Biophysical Chemistry 161,

15 Nelson, D.L., Cox, M. M. (2013) Lehniger Principles of Biochemistry, Sixth Ed. W.H. Freeman and Co: New York Taylor SS, Knighton DR, Zheng J, Sowadski JM, Gibbs CS, Zoller MJ. A template for the protein kinase family. Trends Biochem Sci 1993; 18(3): Taylor, S., Zhang, P., Steichen, J. M., Keshwani, M. M., Kornev, A. P. (2013) PKA: Lessons learned after twenty years. Biochimica et Biophysica Acta 1834, Yonemoto, W., Garred, S. M., Bell, S. M., Taylor, S.S. (1993) Identification of the phosphorylation sites in the recombinant catalytic subunit of camp-dependent Protein Kinase. J. Biol. Chem. 269 (25): Zhou, J.; Adams, J. A. (1997) Is there a catalytic base in the active site of campdependent Protein Kinase? Biochemistry, 36, 2977.

16 Figure Legends Figure 1 The catalytic subunit of PKA is shown in green and the Regulatory subunit is colored blue. When inactive, the regulatory subunit occupies the substratebinding site of the catalytic subunit. PDB ID: 2QCS Figure 2 The substrate-binding site of the PKAc is shown. Both the catalytic (green) and regulatory (blue) subunits are shown as cartoons. Key conserved residues in the active site are highlighted. Ser53 is colored orange and is part of the glycine-rich loop. Lysine 168 is pink. Asp166 is yellow and orients the substrate through hydrogen bonding. Figure 3 Figure was created using Pymol and PDB ID 1ATP. Yellow spheres depict the C- spine in PKA catalytic subunit. Green spheres show the R spine. The F-helix, which runs through the middle of the C-lobe, is shown in red. The F-helix is a foundation for the C and R spines. Figure 4 The cartoon diagram shows PKA (PDB ID: 1ATP). Gray coloring indicates the N- lobe while green coloring indicates the C-lobe of the catalytic subunit of PKA.

17 The cyan alpha helix indicates the A-helix. Phosphorylated Ser338 and Thr197 are shown as sticks. Figure 5 PKAc is shown in a green carton diagram. Magnesium ions are purple spheres. The pink amino acid residue is Lys168; Lys72 is colored orange. In gray, Asp166 is shown. Yellow denotes Ser53. Glycine-rich loop is shown in blue. ATP is shown in a stick representation, black. Figure 6 The catalytic domain of PKA is shown as a cartoon diagram. Conserved subdomains are colored as follows: subdomain I, magenta; subdomain II, yellow; subdomain III, blue; subdomain IV, red; subdomain V, grey; subdomain VI, cyan; subdomain VII, deep teal; subdomain VIII, raspberry; subdomain IX, sand; subdomain X, orange; subdomain XI, forest.

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