Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.     

Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-(32)P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.     

Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate, and specificity of the phosphorylation site sequence in the whole protein

Pyk1 (pyruvate kinase 1) from Saccharomyces cerevisiae was characterized as a substrate for PKA (protein kinase A) from bovine heart and yeast. By designing Pyk1 synthetic peptides containing potential PKA sequence targets (Ser22, Thr94 and Thr478) we determined that the peptide S22 was a substrate for PKA in vitro, with a K(sp)* (specificity constant) 10-fold and 3-fold higher than Kemptide for bovine heart and yeast PKA respectively. In vitro phosphorylation of the Pyk1 S22A mutant protein was decreased by as much as 90% when compared with wild-type Pyk1 and the Pyk1 T94A mutant. The K(sp)* values for Pyk1 and Pyk1 T94A were the same, indicating that both proteins are phosphorylated at the same site by PKA. Two-dimensional PAGE of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation prevented the formation of one of the Pyk1 isoforms. We conclude that in yeast the major PKA phosphorylation site of Pyk1 is Ser22. Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more active in the absence of FBP (fructose 1,6-bisphosphate). The specificity of yeast and mammalian PKA towards the S22 peptide and towards whole Pyk1 protein was measured and compared. The K(sp)* for the S22 peptide is higher than that for Pyk1, indicating that the peptide modelled on Pyk1 is a much better substrate than Pyk1, regardless of which tissue was used as the source of PKA. However, the K(m) of Pyk1 protein is lower than that of the better substrate, the S22 peptide, indicating that ground-state substrate binding is not the major determinant of substrate specificity for PKA.     

The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons.

Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activity of class C L-type Ca(2+) channels which are clustered at postsynaptic sites and are important regulators of neuronal functions. We investigated a possible mechanism that could ensure rapid and efficient phosphorylation of these channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PKA and target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation contain an endogenous kinase that phosphorylates kemptide, a classic PKA substrate peptide, and also the main phosphorylation site for PKA in the pore-forming alpha(1) subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PKA inhibitory peptide PKI(5-24) and stimulated by cAMP. Physical association of the catalytic C subunit of PKA with the immunoisolated class C channel complex was confirmed by immunoblotting. A direct protein overlay binding assay performed with (32)P-labeled RIIbeta revealed a prominent AKAP with an M(r) of 280,000 in class C channel complexes. The protein was identified by immunoblotting as the microtubule-associated protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules. In vitro experiments show that MAP2B can directly bind to the alpha(1) subunit of the class C channel. Our findings indicate that PKA is an integral part of neuronal class C L-type Ca(2+) channels and suggest that the AKAP MAP2B may mediate this interaction. Neither PKA nor MAP2B were detected in immunoprecipitates of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamate receptors or class B N-type Ca(2+) channels. Accordingly, MAP2B docked at class C Ca(2+) channels may be important for recruiting PKA to postsynaptic sites.     

Alteration of a cyclic AMP-dependent protein kinase phosphorylation site in the c-Fos protein augments its transforming potential.

We have studied the phosphorylation of the nuclear oncoprotein Fos by cyclic AMP-dependent protein kinase (PKA). We demonstrate that the human c-Fos protein, phosphorylated either in vitro with purified PKA or in vivo in JEG3 cells following treatment with forskolin, has similar phosphotryptic peptide maps. Serine 362, which constitutes part of a canonical PKA phosphorylation site (RKGSSS), is phosphorylated both in vivo and in vitro. A mutant of Fos protein in which serine residues 362 to 364 have been altered to alanine residues is not efficiently phosphorylated in vitro. Furthermore, Fos protein in which serines 362 to 364 have been altered to alanine shows increased transforming potential. We propose that phosphorylation of Fos by PKA is an important regulatory step in controlling its activity in normal cell growth and differentiation.     

An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin–agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.     

Phosphorylation of membrane-bound acetylcholine receptor by protein kinase C: characterization and subunit specificity

Acetylcholine receptor (AChR) from Torpedo electric organ in its membrane-bound or solubilized form is phosphorylated by the Ca2+/phospholipid-dependent protein kinase (PKC). The subunit specificity for PKC is different from that observed for cAMP-dependent protein kinase (PKA). Whereas PKC phosphorylates predominantly the delta subunit and the phosphorylation of the gamma subunit by this enzyme is very low, PKA phosphorylates both subunits to a similar high extent. We have extended our phosphorylation studies to a synthetic peptide from the gamma subunit, corresponding to residues 346-359, which contains a consensus PKA phosphorylation site. This synthetic peptide is phosphorylated by both PKA and PKC, suggesting that in the intact receptor both kinases may phosphorylate the gamma subunit at a similar site, as has been previously demonstrated by us for the delta subunit [Safran, A., et al. (1987) J. Biol. Chem. 262, 10506-10510]. The diverse pattern of phosphorylation of AChR by PKA and PKC may play a role in the regulation of its function.     

Phosphorylation of cysteine string protein on Serine 10 triggers 14-3-3 protein binding

Cysteine string protein (CSP) is a neuronal chaperone that maintains normal neurotransmitter exocytosis and is essential for preventing presynaptic neurodegeneration. CSP is phosphorylated in vivo on a single residue, Ser10, and this phosphorylation regulates its cellular functions, although the molecular mechanisms involved are unclear. To identify novel phosphorylation-specific binding partners for CSP, we used a pull-down approach using synthetic peptides and recombinant proteins. A single protein band was observed to bind specifically to a Ser10-phosphorylated CSP peptide (residues 4-14) compared to a non-phosphorylated peptide. This band was identified as 14-3-3 protein of various isoforms using mass spectrometry and Western blotting. PKA phosphorylation of full-length CSP protein stimulated 14-3-3 binding, and this was abolished in a Ser10-Ala mutant CSP, confirming the binding site as phospho-Ser10. As both CSP and 14-3-3 proteins are implicated in neurotransmitter exocytosis and neurodegeneration, this novel phosphorylation-dependent interaction may help maintain the functional integrity of the synapse.     

Catalytic subunit of cAMP-dependent protein kinase from a catch muscle of the bivalve mollusk Mytilus galloprovincialis: purification, characterization, and phosphorylation of muscle proteins

cAMP-dependent protein kinase (PKA) plays a crucial role in the release of the catch state of molluskan muscles, but the nature of the enzyme in such tissues is unknown. In this paper, we report the purification of the catalytic (C) subunit of PKA from the posterior adductor muscle (PAM) of the sea mussel Mytilus galloprovincialis. It is a monomeric protein with an apparent molecular mass of 40.0+/-2.0kDa and Stoke's radius 25.1+/-0.3A. The protein kinase activity of the purified enzyme was inhibited by both isoforms of the PKA regulatory (R) subunit that we had previously characterized in the mollusk, and also by the inhibitor peptide PKI(5-24). On the other hand, the main proteins of the contractile apparatus of PAM were partially purified and their ability to be phosphorylated in vitro by purified PKA C subunit was analyzed. The results showed that twitchin, a high molecular mass protein associated with thick filaments, was the better substrate for endogenous PKA. It was rapidly phosphorylated with a stoichiometry of 3.47+/-0.24mol Pmol(-1) protein. Also, catchin, paramyosin, and actin were phosphorylated, although more slowly and to a lesser extent. On the contrary, myosin heavy chain (MHC) and tropomyosin were not phosphorylated under the conditions used.     

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth.     

Determination of a cAMP-dependent protein kinase phosphorylation site in the C-terminal region of human endothelial actin-binding protein

Three different C-terminal regions of human endothelial actin-binding protein-280 (ABP-280 or ABP; nonmuscle filamin) were subcloned and efficiently expressed in the Escherichia coli BL21 (DE3) system as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As predicted by the aminoacid sequence one of the fragments, a 109-kDa peptide (residues 1671-2647), contained a calpain cleavage site and two potential cAMP-dependent protein kinase (PKA) phosphorylation sites (serine 2152 and threonine 2336). A second fragment, a 74-kDa peptide (residues 1671-2331), contained a calpain cleavage site and one of the three presumptive PKA phosphorylation sites (serine 2152). The third fragment, a 48-kDa peptide (residues 2223-2647), contained only one of the PKA sites (threonine 2336). Phosphorylation of these truncated peptides indicated that only the fragments containing serine 2152 incorporated phosphate after PKA treatment. Site-directed mutagenesis analysis confirmed that serine 2152 is the unique substrate for PKA in the C-terminal region of ABP. The functional significance of phosphorylation of this residue, which belongs to a serine-proline motif, is discussed.     

Removal of DnaK contamination during fusion protein purifications

The use of fusion proteins for recombinant protein expression in Escherichia coli has become popular because the carrier increases protein solubility, standardizes expression levels, and facilitates purification of the fusion products. However, we have observed that the peptide regions that fuse the carrier to the protein of interest bind E. coli Hsp70 molecular chaperones (DnaK) depending on their amino acid composition, resulting in an unwanted contamination during protein purification. Here we describe an approach that helps to circumvent this unwanted contamination. First, the appropriate amino acids surrounding and comprising the cloning site are chosen by using a software based on an algorithm already developed to decrease to a minimum the propensity of the fusion protein to bind DnaK. Second, DnaK contamination is significantly reduced by washing the fusion protein bound to the purification resin with MgATP plus soluble denatured E. coli proteins before elution. The approach can also be applied to eliminate other molecular chaperones.     

Folding and activity of cAMP-dependent protein kinase mutants

The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1H, 15N-TROSY NMR experiments.     

The cAMP-dependent protein kinase phosphorylates the rap1 protein in vitro as well as in intact fibroblasts, but not the closely related rap2 protein

The products of rap genes (rap1A, rap1B and rap2) are small molecular weight GTP-binding proteins that share approximately 50% homology with ras-p21s. It had previously been shown that a rap1 protein (also named Krev-1 or smg p21) could be phosphorylated on serine residues by the cAMP-dependent protein kinase (PKA) in vitro as well as in intact platelets stimulated by prostaglandin E1. We show here that the rap1A protein purified from recombinant bacteria is phosphorylated in vitro by the catalytic subunit of PKA and that the deletion of the 17 C-terminal amino acids leads to the loss of this phosphorylation. This suggests that the serine residue at position 180 constitutes the site of phosphorylation of the rap1A protein by PKA. The rap1 protein can also be phosphorylated by PKA in intact fibroblasts; this phenomenon is independent of their proliferative state. In contrast, protein kinase C (PKC) does not phosphorylate the rap1 proteins, neither in vitro nor in vivo. Finally, the 60% homologous rap2 protein is neither phosphorylated in vitro nor in vivo by PKA or PKC.     

Phosphorylation of RII subunit and attenuation of cAMP-dependent protein kinase activity by proline-directed protein kinase.

Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.7 mol phosphate/mol RII subunit protein. The major RII phosphorylation site is identified as a threonine residue located within a large hydrophobic tryptic peptide that is predicted to contain the cAMP binding domains. In contrast to the reported effects of RII autophosphorylation, kinetic analysis of RII function following phosphorylation by PDPK indicates that the inhibitory potency of RII toward the catalytic subunit of PKA in a reassociation assay is increased in proportion to the degree of phosphorylation. Further studies indicate that the cAMP-dependent activation of the RII2C2 holoenzyme is inhibited by PDPK phosphorylation. Taken together, the results of these studies indicate that phosphorylation of RII by PDPK attenuates the activity of PKA. This antagonistic interaction suggests a biochemical mechanism by which a growth factor-activated signaling system may function to modulate cAMP-dependent cellular responses.     

Bicarbonate-induced phosphorylation of p270 protein in mouse sperm by cAMP-dependent protein kinase

Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility.     

Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14-18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.