Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.     

Structural and functional analysis of the cAMP binding domain from the regulatory subunit of Mucor rouxii protein kinase A

The cAMP binding domain of the regulatory subunit (R) of Mucor rouxii protein kinase A was cloned. The deduced amino acid sequence was highly homologous in sequence and in size to the corresponding region in fungal and higher eukaryotic regulatory subunits (47-54%), but particularly homologous (62%) to Blastocladiella emersonii, a fungus classified in a different phylum. Amino acids reported to be important for interaction with cAMP, for cooperativity between the two cAMP binding domains, in the general folding of the domain, and for interaction with the catalytic subunit were conserved in all the fungal sequences. Based on either sequence or functional behavior, the M. rouxii R subunit cannot be classified as being more similar to RI or RII of mammalian systems. The M. rouxii protein sequence was modeled using as template the coordinates of the crystallized bovine regulatory subunit type Ialpha. The quality of the model is good. The two backbones could be perfectly overlapped, except for two loop regions of high divergence. The alpha helix C of domain A, proposed to have a strong interaction with the catalytic subunit, contains a leucine replacing a basic residue (arginine or lysine) commonly found in RI or RII. The domains A and B of the M. rouxii regulatory subunit were overexpressed as fusion proteins with GST. GST domain B protein was inactive. GST domain A was active; the kinetic parameters of affinity toward cAMP analogs, site selectivity, and dissociation kinetics of bound cAMP were analogous to the properties of the domain in the whole regulatory subunit.     

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.     

Probing cAMP-dependent protein kinase holoenzyme complexes I alpha and II beta by FT-IR and chemical protein footprinting

Although individual structures of cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits have been determined at the atomic level, our understanding of the effects of cAMP activation on protein dynamics and intersubunit communication of PKA holoenzymes is very limited. To delineate the mechanism of PKA activation and structural differences between type I and II PKA holoenzymes, the conformation and structural dynamics of PKA holoenzymes Ialpha and IIbeta were probed by amide hydrogen-deuterium exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and chemical protein footprinting. Binding of cAMP to PKA holoenzymes Ialpha and IIbeta leads to a downshift in the wavenumber for both the alpha-helix and beta-strand bands, suggesting that R and C subunits become overall more dynamic in the holoenzyme complexes. This is consistent with the H-D exchange results showing a small change in the overall rate of exchange in response to the binding of cAMP to both PKA holoenzymes Ialpha and IIbeta. Despite the overall similarity, significant differences in the change of FT-IR spectra in response to the binding of cAMP were observed between PKA holoenzymes Ialpha and IIbeta. Activation of PKA holoenzyme Ialpha led to more conformational changes in beta-strand structures, while cAMP induced more apparent changes in the alpha-helical structures in PKA holoenzyme IIbeta. Chemical protein footprinting experiments revealed an extended docking surface for the R subunits on the C subunit. Although the overall subunit interfaces appeared to be similar for PKA holoenzymes Ialpha and IIbeta, a region around the active site cleft of the C subunit was more protected in PKA holoenzyme Ialpha than in PKA holoenzyme IIbeta. These results suggest that the C subunit assumes a more open conformation in PKA holoenzyme IIbeta. In addition, the chemical cleavage patterns around the active site cleft of the C subunit were distinctly different in PKA holoenzymes Ialpha and IIbeta even in the presence of cAMP. These observations provide direct evidence that the R subunits may be partially associated with the C subunit with the pseudosubstrate sequence docked in the active site cleft in the presence of cAMP.     

Proteolytic regulation of the mitochondrial cAMP-dependent protein kinase

The mitochondrial cAMP-dependent protein kinase (PKA) is activatable in a cAMP-independent fashion. The regulatory (R) subunits of the PKA holoenzyme (R(2)C(2)), but not the catalytic (C) subunits, suffer proteolysis upon exposure of bovine heart mitochondria to digitonin, Ca(2+), and a myriad of electron transport inhibitors. Selective loss of both the RI- and RII-type subunits was demonstrated via Western blot analysis, and activation of the C subunit was revealed by phosphorylation of a validated PKA peptide substrate. Selective proteolysis transpires in a calpain-dependent fashion as demonstrated by exposure of the R and C subunits of PKA to calpain and by attenuation of R and C subunit proteolysis in the presence of calpain inhibitor I. By contrast, exposure of mitochondria to cAMP fails to promote R subunit degradation, although it does result in enhanced C subunit catalytic activity. Treatment of mitochondria with electron transport chain inhibitors rotenone, antimycin A, sodium azide, and oligomycin, as well as an uncoupler of oxidative phosphorylation, also elicits enhanced C subunit activity. These results are consistent with the notion that signals, originating from cAMP-independent sources, elicit enhanced mitochondrial PKA activity.     

The conformationally dynamic C helix of the RIalpha subunit of protein kinase A mediates isoform-specific domain reorganization upon C subunit binding

Different isoforms of the full-length protein kinase A (PKA) regulatory subunit homodimer (R2) and the catalytic (C) subunit-bound holoenzyme (R2C2) have very different global structures despite similar molecular weights and domain organization within their primary sequences. To date, it has been the linker sequence between the R subunit dimerization/docking domain and cAMP-binding domain A that has been implicated in modulating domain interactions to give rise to these differences in global structure. The small angle solution scattering data presented here for three different isoforms of PKA heterodimer (deltaR-C) complexes reveal a role for another conformationally dynamic sequence in modulating inter-subunit and domain interactions, the C helix that connects the cAMP-binding domains A and B of the R subunit. The deltaR-C heterodimer complexes studied here were each formed with a monomeric N-terminal deletion mutant of the R subunit (deltaR) that contains the inhibitor sequence and both cAMP-binding domains. The scattering data show that type IIalpha and type IIbeta deltaR-C heterodimers are relatively compact and globular, with the C subunit contacting the inhibitor sequence and both cAMP-binding domains. In contrast, the type Ialpha heterodimer is significantly more extended, with the C subunit interacting with the inhibitor sequence and cAMP-binding domain A, whereas domain B extends out such that its surface is almost completely solvent exposed. These data implicate the C helix of RIalpha in modulating isoform-specific interdomain communication in the PKA holoenzyme, adding another layer of structural complexity to our understanding of signaling dynamics in this multisubunit, multidomain protein kinase.     

Critical role of the N-terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes

A putative PEST sequence was recently identified close to the N-terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes. The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence. Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L. monocytogenes. Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif. The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering). Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence. Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial -virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L. -monocytogenes.     

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.     

Characterization of the regulatory subunit of Yarrowia lipolytica cAMP-dependent protein kinase. Evidences of a monomeric protein

cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.     

Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.     

Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA

cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.     

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.     

Presence of AMP binding sequence in subunit B of Corynebacterium sarcosine oxidase

Corynebacterium sarcosine oxidase is composed of A, B, C, and D subunits. To characterize these subunits, we analyzed their N-terminal sequences by automated Edman degradation. We identified 20 residues of subunit A, 58 of B, 31 of C, and 33 of D. There was no homology among these sequences according to secondary structure predictions and hydrophilicity profiles. But we found that subunit B contained a sequence homologous to that of the AMP-binding site of other flavoproteins.     

Purification and characterization of cAMP dependent protein kinase from Microsporum gypseum

A cyclic AMP dependent protein kinase (PKA), its regulatory (R) and catalytic (C) subunits were purified to homogeneity from soluble extract of Microsporum gypseum. Purified enzyme showed a final specific activity of 277.9 nmol phosphate transferred min(-1) mg protein(-1) with kemptide as substrate. The enzyme preparation showed two bands with molecular masses of 76 kDa and 45 kDa on sodium dodecyl polyacrylamide gel electrophoresis. The 76 kDa subunit was found to be the regulatory (R) subunit of PKA holoenzyme as determined by its immunoreactivity and the isoelectric point of this subunit was 3.98. The 45 kDa subunit was found to be the catalytic (C) subunit by its immunoreactivity and phosphotransferase activity. Gel filtration using Sepharose CL-6B revealed the molecular mass of PKA holoenzyme to be 240 kDa, compatible with its tetrameric structure, consisting of two regulatory subunits (76 kDa) and two catalytic subunits (45 kDa). The specificity of enzyme towards protein acceptors in decreasing order of phosphorylation was found to be kemptide, casein, syntide and histone IIs. Purified enzyme had apparent K(m) values of 71 microM and 25 microM for ATP and kemptide, respectively. Phosphorylation was strongly inhibited by mammalian PKA inhibitor (PKI) but not by inhibitors of other protein kinases. The PKA showed maximum activity at pH 7.0 and enzyme activity was inhibited in the presence of N-ethylmaleimide (NEM) which shows the involvement of sulfhydryl groups for the activity of PKA. PKA phosphorylated a number of endogenous proteins suggesting the multifunctional role of cAMP dependent protein kinase in M. gypseum. Further work is under progress to identify the natural substrates of this enzyme through which it may regulate the enzymes involved in phospholipid metabolism.     

Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency

Protease C1, an enzyme from soybean (Glycine max [L.] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the alpha' and alpha subunits, but not the beta subunit, of beta-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the alpha' and alpha subunits of beta-conglycinin may be located in their N-terminal domain, which is not found in the beta subunit of beta-conglycinin. To check this hypothesis, storage proteins from other plant species that are homologous to either the alpha'/alpha or the beta subunit of beta-conglycinin were tested as substrates. As expected, the convicilin from pea (Pisum sativum), a protein homologous to the alpha' and alpha subunits of beta-conglycinin, was digested by protease C1. The vicilins from pea as well as vicilins from adzuki bean (Vigna angularis), garden bean (Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), and mung bean (Vigna radiata), storage proteins that are homologous to the beta subunit of soybean beta-conglycinin, were not degraded by protease C1. Degradation of soybean beta-conglycinin involves a sequential attack of the alpha subunit at multiple sites, culminating in the formation of a stable intermediate of 53.5 kD and a final product of 48.0 kD. The cleavage sites resulting in this formation of the intermediates and final product were determined by N-terminal analysis. These were compared to the known amino acid sequences of the three beta-conglycinin subunits. Results showed these two polypeptides to be generated by proteolysis of the alpha subunit at regions bearing long strings of acidic amino acid residues.     

Attenuation of green fluorescent protein half-life in mammalian cells

The half-life of the green fluorescent protein (GFP) was determined biochemically in cultured mouse LA-9 cells. The wild-type protein was found to be stable with a half-life of approximately 26 h, but could be destabilized by the addition of putative proteolytic signal sequences derived from proteins with shorter half-lives. A C-terminal fusion of a PEST sequence from the mouse ornithine decarboxylase gene reduced the half-life to 9.8 h, resulting in a GFP variant suitable for the study of dynamic cellular processes. In an N-terminal fusion containing the mouse cyclin B1 destruction box, it was reduced to 5.8 h, with most degradation taking place at metaphase. The combination of both sequences produced a similar GFP half-life of 5.5 h. Thus, the stability of this marker protein can be controlled in predetermined ways by addition of the appropriate proteolytic signals.     

Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.