Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.     

Protein kinase C phosphorylation of purified Na,K-ATPase: C-terminal phosphorylation sites at the alpha- and gamma-subunits close to the inner face of the plasma membrane

The alpha-subunit of the Na,K-ATPase is phosphorylated at specific sites by protein kinases A and C. Phosphorylation by protein kinase C (PKC) is restricted to the N terminus and takes place to a low stoichiometry, except in rat. Here we show that the alpha-subunit of shark Na,K-ATPase can be phosphorylated by PKC at C-terminal sites to stoichiometric levels in the presence of detergents. Two novel phosphorylation sites are possible candidates for this PKC phosphorylation: Thr-938 in the M8/M9 loop located very close to the PKA site, and Ser-774, in the proximal part of the M5/M6 hairpin. Both sites are highly conserved in all known alpha-subunits, indicating a physiological role. A similar pattern of detergent-mediated phosphorylation by PKC was found in pig kidney Na,K-ATPase alpha-subunit. Interestingly, the kidney-specific gamma-subunit was phosphorylated by PKC in the presence of detergent. The close proximity of the novel PKC sites to the membrane suggests that targeting proteins to tether PKC into the membrane phase is important in controlling the in vivo phosphorylation of this novel class of membrane-adjacent PKC sites. It is suggested that in purified preparations where functional targeting may be impaired detergents are needed to expose the sites.     

Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase c via a novel member of the FXYDY family

The Na,K-ATPase provides the driving force for many ion transport processes through control of Na(+) and K(+) concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, alpha and beta. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the gamma-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the gamma-subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na, K-ATPase alpha-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the alpha-subunit of pig kidney Na, K-ATPase, indicating specific association with different alpha-isoforms. Finally, PLMS and the alpha-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C(12)E(8) resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.     

Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases.

Phospholemman (PLM), the major sarcolemmal substrate for phosphorylation by cAMP-dependent kinase (PKA) protein kinase C (PKC) and NIMA kinase in muscle, induces hyperpolarization-activated anion currents in Xenopus oocytes, most probably by enhancing endogenous oocyte currents. PLM peptides from the cytoplasmic tail are phosphorylated by PKA at S68, by NIMA kinase at S63, and by PKC at both S63 and S68. We have confirmed the phosphorylation sites in the intact protein, and we have investigated the role of phosphorylation in the regulatory activity of PLM using oocyte expression experiments. We found: (1) the cytoplasmic domain is not essential for inducing currents in oocytes; (2) co-expression of PKA increased the amplitude of oocyte currents and the amount of PLM in the oocyte membrane largely, but not exclusively, through phosphorylation of S68; (3) co-expression of PKA had no effect on a PLM mutant in which all putative phosphorylation sites had been inactivated by serine to alanine mutation (SSST 62, 63, 68, 69 AAAA); (4) co-expression of PKC had no effect in this system; (5) co-expression of NIMA kinase increased current amplitude and membrane protein level, but did not require PLM phosphorylation. These findings point to a role for phosphorylation in the function of PLM.     

Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.     

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (11-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol P_i/mol of -subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P_i into the -subunit and the loss of activity of the Na,K-ATPase.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4beta2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated with ATP and either PKA or PKC. Both alpha4 and alpha4(336-597) were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the alpha4(336-597) fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and alpha4(336-597) were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amino acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP and the kinases. The phosphorylation of alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 microM and 160.8 +/- 26.8 microM, respectively, suggesting that Ser(368) was a better substrate for PKA than PKC.     

Functional significance of the shark Na,K-ATPase N-terminal domain. Is the structurally variable N-Terminus involved in tissue-specific regulation by FXYD proteins?

The proteolytic profile after mild controlled trypsin cleavage of shark rectal gland Na,K-ATPase was characterized and compared to that of pig kidney Na,K-ATPase, and conditions for achieving N-terminal cleavage of the alpha-subunit at the T(2) trypsin cleavage site were established. Using such conditions, the shark enzyme N-terminus was much more susceptible to proteolysis than the pig enzyme. Nevertheless, the maximum hydrolytic activity was almost unaffected for the shark enzyme, whereas it was significantly decreased for the pig kidney enzyme. The apparent ATP affinity was unchanged for shark but increased for pig enzyme after N-terminal truncation. The main common effect following N-terminal truncation of shark and pig Na,K-ATPase is a shift in the E(1)-E(2) conformational equilibrium toward E(1). The phosphorylation and the main rate-limiting E(2) --> E(1) step are both accelerated after N-terminal truncation of the shark enzyme, but decreased significantly in the pig kidney enzyme. Some of the kinetic differences, like the acceleration of the phosphorylation reaction, following N-terminal truncation of the two preparations may be due to the fact that under the conditions used for N-terminal truncation, the C-terminal domain of the FXYD regulatory protein of the shark enzyme, PLMS or FXYD10, was also cleaved, whereas the gamma or FXYD2 of the pig enzyme was not. In the shark enzyme, N-terminal truncation of the alpha-subunit abolished association of exogenous PLMS with the alpha-subunit and the functional interactions were abrogated. Moreover, PKC phosphorylation of the preparation, which relieves PLMS inhibition of Na,K-ATPase activity, exposed the N-terminal trypsin cleavage site. It is suggested that PLMS interacts functionally with the N-terminus of the shark Na,K-ATPase to control the E(1)-E(2) conformational transition of the enzyme and that such interactions may be controlled by regulatory protein kinase phosphorylation of the N-terminus. Such interactions are likely in shark enzyme where PLMS has been demonstrated by cross-linking to associate with the Na,K-ATPase A-domain.     

Phosphorylation of the Na,K-ATPase by Ca,phospholipid-dependent andcAMP-dependent protein kinases. Mapping of the region phosphorylated by Ca,phospholipid-dependent protein kinase

Ca,phospholipid-dependent (PKC) andcAMP-dependent (PKA) protein kinases phosphorylate the -subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol³²P/mol of -subunit, respectively. PKA (in contrast to PKC) phosphorylates the -subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that³²P is incorporated into the N-terminal 41-kDa fragment of the -subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the -subunit molecule. These findings suggest that PKC phosphorylates the -subunit of the Na,K-ATPase within the region restricted by C₃ and T₁ cleavage sites.     

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.     

Phosphorylation and activation of brain aromatic L-amino acid decarboxylase by cyclic AMP-dependent protein kinase

Aromatic L-amino acid decarboxylase (AAAD), an enzyme required for the synthesis of catecholamines, indoleamines, and trace amines, is rapidly activated by cyclic AMP-dependent pathways in striatum and midbrain in vivo, suggesting enzyme phosphorylation. We now report that the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) directly phosphorylated AAAD immunoprecipitated from homogenates prepared from the mouse striatum and midbrain in vitro. Under the same phosphorylation conditions, the catalytic subunit of PKA also phosphorylated a recombinant AAAD protein expressed in Escherichia coli transfected with an AAAD cDNA isolated from the bovine adrenal gland. The PKA-induced AAAD phosphorylation of immunoprecipitates from striatum and midbrain was time and concentration dependent and blocked by a specific PKA peptide inhibitor. Incubation of the catalytic subunit of PKA with striatal homogenates increased enzyme activity by approximately 20% in a time- and concentration-dependent manner. Moreover, incubation of the catalytic subunit of PKA with recombinant AAAD increased activity by approximately 70%. A direct phosphorylation of AAAD protein by PKA might underlie the cyclic AMP-induced rapid and transient activation of AAAD in vivo.     

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 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.     

Interaction of protein kinase C and cAMP-dependent pathways in the phosphorylation of the Na,K-ATPase

To test the hypothesis that there is cross-talk between the protein kinase C (PKC) and protein kinase A (PKA) pathways in the regulation of the Na,K-ATPase, we measured its phosphorylation in mammalian cell cultures. Phosphorylation of the PKC site, Ser-18, appeared to be due to the activation of the alpha isoform of the kinase. In NRK-52E and L6 cells, this phosphorylation was reduced by prior activation of a cAMP-dependent signaling pathway with forskolin. In principle this would be consistent with direct interaction between the two phosphorylation sites, but further investigation suggested a more indirect mechanism. First, phosphorylation of Ser-938, the PKA site, could not be detected despite the presence of active PKA. Second, there was a major reduction in the phosphorylation of unrelated phosphoproteins as a consequence of elevation of cAMP, suggesting generalized reduction of kinase activity or activation of phosphatase activity. In NRK-52E and L6, phosphorylation of the Na, K-ATPase at Ser-18 paralleled this global change. In C6 cells, in contrast, there was no cAMP effect on Na,K-ATPase phosphorylation at Ser-18 and no global cAMP effect on other phosphoproteins. The cross-talk is evidently mediated by events occurring at the cellular level.     

Ordered multisite phosphorylation of Xenopus ribosomal protein S6 by S6 kinase II.

Phosphorylated ribosomal proteins were isolated from Xenopus 40 S ribosomal subunits by reversed-phase high performance liquid chromatography (HPLC) to enable direct analysis of the phosphorylation sites in ribosomal protein S6. Xenopus S6 closely resembled mammalian S6 with respect to the following properties: (i) reversed-phase HPLC elution behavior, (ii) amino-terminal sequence (96% identity in the first 37 residues), and (iii) an identical sequence within the region of its phosphorylation sites. Whereas S6 was the only ribosomal protein phosphorylated in vitro by Xenopus S6 kinase II, ribosomes phosphorylated in vivo were found to be associated with an additional phosphoprotein having an amino-terminal sequence identical to that of the ubiquitin carboxyl-terminal extension protein CEP 80. S6 kinase II phosphorylated at least four sites (serines 1-3 and 5) in the sequence Arg-Arg-Leu-Ser(1)-Ser(2)-Leu-Arg-Ala-Ser(3)-Thr-Ser(4)-Lys-Ser(5)-, which correspond to the residues known to be phosphorylated in the carboxyl-terminal region of mammalian S6. The in vivo S6 phosphorylation sites in maturing Xenopus oocytes were shown to be located within the same cluster of serine residues, although individual sites were not identified. Kinetic analysis of S6 kinase II-catalyzed phosphorylation events indicated a simple sequential mechanism of multisite phosphorylation initiating at either serine 2 (preferred) or serine 1, with the rates of phosphorylation of individual sites occurring in the order serine 2 greater than serine 1 greater than serine 3 greater than serine 5.     

A purified S6 kinase kinase from Xenopus eggs activates S6 kinase II and autophosphorylates on serine, threonine, and tyrosine residues.

S6 kinases I and II have been purified previously from Xenopus eggs and shown to be activated by phosphorylation on serine and threonine residues. An S6 kinase clone, closely related to S6 kinase II, was subsequently identified and the protein product was expressed in a baculovirus system. Using this protein, termed "rsk" for Ribosomal Protein S6 Kinase, as a substrate, we have purified to homogeneity from unfertilized Xenopus eggs a 41-kDa serine/threonine kinase termed rsk kinase. Both microtubule-associated protein-2 and myelin basic protein are good substrates for rsk kinase, whereas alpha-casein, histone H1, protamine, and phosvitin are not. rsk kinase is inhibited by low concentrations of heparin as well as by beta-glycerophosphate and calcium. Activation of rsk kinase during Xenopus oocyte maturation is correlated with phosphorylation on threonine and tyrosine residues. However, in vitro, rsk kinase undergoes autophosphorylation on serine, threonine, and tyrosine residues, identifying it as a "dual specificity" enzyme. Purified rsk kinase can be inactivated in vitro by either a 37-kDa T-cell protein-tyrosine phosphatase or the serine/threonine protein phosphatase 2A. Phosphatase-treated S6KII can be reactivated by rsk kinase, and S6 kinase activity in resting oocyte extracts increases significantly when purified rsk kinase is added. The availability of purified rsk kinase will enhance study of the signal transduction pathway(s) regulating phosphorylation of ribosomal protein S6 in Xenopus oocytes.     

Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C

Rho-associated kinase (Rho-kinase/ROCK/ROK) is a serine/threonine kinase and plays an important role in various cellular functions. The cAMP-dependent protein kinase (protein kinase A/PKA) and protein kinase C (PKC) are also serine/threonine kinases, and directly and/or indirectly take part in the signal transduction pathways of Rho-kinase. They have similar phosphorylation site motifs, RXXS/T and RXS/T. The purpose of this study was to identify whether sites phosphorylated by Rho-kinase could be targets for PKA and PKC and to find peptide substrates that are specific to Rho-kinase, i.e., with no phosphorylation by PKA and PKC. A total of 18 substrates for Rho-kinase were tested for phosphorylation by PKA and PKC. Twelve of these sites were easily phosphorylated. These results mean that Rho-kinase substrates can be good substrates for PKA and/or PKC. On the other hand, six Rho-kinase substrates showing no or very low phosphorylation efficiency (<20%) for PKA and PKC were identified. Kinetic parameters (K(m) and k(cat)) showed that two of these peptides could be useful as substrates specific to Rho-kinase phosphorylation.     

Phosphorylation of the alpha-subunits of the Na+/K+-ATPase from mammalian kidneys and Xenopus oocytes by cGMP-dependent protein kinase results in stimulation of ATPase activity.

Phosphorylation of Na+/K+-ATPase by cGMP-dependent protein kinase (PKG) has been studied in enzymes purified from pig, dog, sheep and rat kidneys, and in Xenopus oocytes. PKG phosphorylates the alpha-subunits of all animal species investigated. Phosphorylation of the beta-subunit was not observed. The stoichiometry of phosphorylation estimated for pig, sheep and dog renal Na+/K+-ATPase is 3.5, 2.2 and 2.1 mol Pi per mol alpha-subunit, respectively. Proteolytic fingerprinting of the pig alpha1-subunits phosphorylated by PKG using specific antibodies raised against N-terminus or C-terminus reveals that phosphorylation sites are located within the intracellular loop of the alpha-subunit between the 35 kDa N-terminal and 27 kDa C-terminal fragments. Phosphorylation sites within the alpha1-subunit of the purified Na+/K+-ATPase do not appear to be easily accessible for PKG since incorporation of Pi requires 0.2% of Triton X-100. Administration of cGMP and PKG in the presence of 5 mm ATP, which prevents inactivation of the Na+/K+-ATPase by detergent, leads to stimulation of hydrolytic activity by 61%. Administration of 50 microm of cGMP or dbcGMP in yolk-free homogenates of Xenopus oocytes leads to stimulation of ouabain-dependent ATPase activity by 130-198% and to incorporation of 33P into the alpha-subunit without the detergent. Hence, PKG plays regulatory role in active transmembraneous transport of Na+ and K+ via phosphorylation of the catalytic subunit of the Na+/K+-ATPase.     

Phosphorylation of laminin-1 by protein kinase C

Several extracellular proteins have been reported to be phosphorylated. Previous studies of our laboratory indicated that laminin-1 can be phosphorylated by protein kinase A (PKA). Moreover, it has been reported that protein kinase C (PKC), although known to be intracellular, can phosphorylate extracellular proteins in the case of cellular damage and/or platelet activation. In the present study we examined the possibility of laminin-1 serving as a substrate of PKC. Amino acid analysis revealed that laminin-1 is phosphorylated by this enzyme on serine residues. Self assembly, heparin binding, and cell attachment on the phosphorylated molecule were then studied. Phosphorylated laminin-1 showed an increased and more rapid self assembly than the non-phosphorylated molecule. Heparin binding and cell attachment experiments indicated enhanced heparin and cell binding capacity of the phosphorylated molecule in comparison to the non- phosphorylated control. These results indicate that laminin-1 can be phosphorylated by protein kinase C. Furthermore, phosphorylation by protein kinase C seems to alter several properties of the molecule, though, the in vivo significance of this phenomenon remains to be studied.