Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. phosphorylation-dependent and -independent mechanisms

We recently identified a region within the cytoplasmic C-terminal tail of the Na+/H+ exchanger NHE3 isoform (residues 579 to 684) which is essential for inhibition of transport activity by cAMP-dependent protein kinase (PKA) (Cabado, A. G., Yu, F. H., Kapus, A., Gergely, L., Grinstein, S., and Orlowski, J. (1996) J. Biol. Chem. 271, 3590-3599). To further define determinants of PKA regulation, six serine residues located in potential recognition sequences for PKA within, or adjacent to, this region (positions 552, 605, 634, 661, 690, and 691) were altered either independently or in various combinations using site-directed mutagenesis. Wild type and mutant NHE3s tagged with the influenza virus hemagglutinin epitope were stably expressed in exchanger-deficient Chinese hamster ovary cells (AP-1) for functional studies. Of the individual mutations examined, only substitutions at Ser605 or Ser634 affected sensitivity to forskolin, an activator of adenylate cyclase, although partial inhibition of NHE3 activity by forskolin remained. By contrast, simultaneous mutation of both these serines completely abolished cAMP-mediated inhibition of NHE3 without greatly affecting basal transport activity. Two-dimensional analysis of tryptic digests of immunoprecipitated NHE3 labeled in vivo with [32P]orthophosphate revealed several phosphopeptides under basal conditions. Phosphorylation was increased approximately 3-fold in one of these peptides following forskolin treatment, and this change was eliminated by mutation of residue Ser605. Thus, phosphorylation of Ser605 is essential for cAMP-mediated inhibition of NHE3. In addition, Ser634 is also required for the effect of cAMP, even though this residue does not become phosphorylated upon activation of PKA.     

Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.     

The role of NHERF and E3KARP in the cAMP-mediated inhibition of NHE3

NHE3 is the apically located Na+/H+ exchanger in the gut and in the renal proximal tubule. Acute inhibition of this transporter by cAMP requires the presence of either of two NHE3-associated proteins, NHERF or E3KARP. It has been suggested that these proteins either directly regulate NHE3 activity after being phosphorylated by protein kinase A (PKA) or that they may serve as adapters that localize PKA near NHE3. We studied the role of NHERF and E3KARP in opossum kidney cells, which endogenously express NHE3, NHERF, and ezrin and display cAMP-dependent inhibition of NHE3. In vivo phosphorylation studies showed that NHERF is a phosphoprotein under basal conditions, but does not change its phosphorylation state after 8-bromo-cAMP treatment, and that E3KARP is not phosphorylated at all. Co-immunoprecipitation showed that NHERF and E3KARP bind both NHE3 and ezrin. Using cAMP analogs it was demonstrated that NHE3 activity, measured as sodium-dependent recovery of the intracellular pH after intracellular acidification, is inhibited by PKA type II. Because others have shown that ezrin binds PKA type II and that NHE3 is phosphorylated by PKA we suggest that NHERF and E3KARP are adapters that link NHE3 to ezrin, thereby localizing PKA near NHE3 to allow NHE3 phosphorylation.     

Differential regulation of cardiac actomyosin S-1 MgATPase by protein kinase C isozyme-specific phosphorylation of specific sites in cardiac troponin I and its phosphorylation site mutants

The significance of site-specific phosphorylation by protein kinase C (PKC) isozymes alpha and delta and protein kinase A (PKA) of troponin I (TnI) and its phosphorylation site mutants in the regulation of Ca(2+)-stimulated MgATPase activity of reconstituted actomyosin S-1 was investigated. The genetically defined TnI mutants used were T144A, S43A/S45A, S43A/S45A/T144A (in which the PKC phosphorylation sites Thr-144 and Ser-43/Ser-45 were respectively substituted by Ala) and N32 (in which the first 32 amino acids in the NH2-terminal sequence containing Ser-23/Ser-24 were deleted). Although the PKC isozymes displayed different substrate phosphorylation kinetics, PKC-alpha phosphorylated equally well TnI wild type and all mutants, whereas N32 was a much poorer substrate for PKC-delta. Furthermore, the two PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in TnI and its mutants, either as individual subunits or as components of the reconstituted troponin complex. Unlike PKC-alpha, PKC-delta favorably phosphorylated the PKA-preferred site Ser-23/Ser-24 and hence, like PKA, reduced the Ca2+ sensitivity of the reconstituted actomyosin S-1 MgATPase. In contrast, PKC-alpha preferred to phosphorylate Ser-43/Ser-45 (common sites for all isozymes) and thus reduced the maximal Ca(2+)-stimulated activity of the MgATPase. In this respect, PKC-delta, by cross-phosphorylating the PKA sites, functioned as a hybrid of PKC-alpha and PKA. The site specificities and hence functional differences between PKC-alpha and -delta were most evident at low phosphorylation (1 mol of phosphate/mol) of TnI wild type and were magnified when S43A/S45A and N32 were used as substrates. The present study has demonstrated, for the first time, that distinct functional consequences could arise from the site-selective preferences of PKC-alpha and -delta for phosphorylating a single substrate in the myocardium, i.e., TnI.     

cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism.     

Spinal nitric oxide mediates antinociception from intravenous morphine

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of 32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser --> Ala mutations in residues 563 and 565 (S563A, S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675 phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.     

Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47(PHOX) S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47(PHOX) as compared with the same cells expressing wild type p47(PHOX). In addition, these mutant p47(PHOX) proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.     

Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms

The majority of familial Alzheimer's disease mutations are linked to the recently cloned presenilin (PS) genes, which encode two highly homologous proteins (PS-1 and PS-2). Full-length PS proteins undergo endoproteolytic cleavage within their hydrophilic loop domain resulting in the formation of C-terminal (CTF) and N-terminal fragments (NTF). PS-2 was found to be phosphorylated as a full-length protein within its N-terminal domain. In contrast, PS-1 is phosphorylated selectively after proteolytic processing within its approximately 20 kDa CTF involving protein kinase C (PKC) and/or protein kinase A (PKA). We now have found that the CTF of the highly homologous PS-2 is also phosphorylated. Surprisingly, the PS-2 CTF is not phosphorylated by PKC or PKA. Instead, the PS-2 CTF is constitutively phosphorylated in vivo by serine/threonine protein kinases, which are independent of phorbol ester and intracellular cAMP. In vitro the large hydrophilic loop of PS-2 between transmembrane domains 6 and 7 can be phosphorylated by casein kinase-1 (CK-1) and CK-2, but not by PKA or PKC. Quantitative analysis of in vitro phosphorylation demonstrates the presence of two phosphorylation sites for CK-1 and a single site for CK-2. A deletion analysis revealed that the CTF of PS-2 is phosphorylated in vivo within an acidic sequence containing three potential phosphorylation sites for CKs (serines 327, 330, and 335). These data suggest that CK type protein kinases phosphorylate the CTF of PS-2 within its hydrophilic loop domain in vivo. Interestingly, the potential phosphorylation sites are located directly adjacent to the recently identified caspase cleavage sites.     

Differential regulation of formyl peptide and platelet-activating factor receptors. Role of phospholipase Cbeta3 phosphorylation by protein kinase A

Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx-insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCbeta3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCbeta3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCbeta3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCbeta3 and appears to block the ability of Gbetagamma to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCbeta3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins.     

Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation

A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M.(1994) J. Biol. Chem. 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation. The PKA-dependent incorporation of phosphate in the partially purified, recombinant rat PDE4D3 followed a time course similar to that of activation. Half-maximal activation of the enzyme was obtained with 0.6 microM ATP and 30 nM of the catalytic subunit of PKA. Phosphorylation altered the Vmax of the PDE without affecting the Km for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram. Cyanogen bromide cleavage of the 32P-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell-free system. Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of 32P was reduced to the largest extent in mutants with both Ser13 --> Ala and Ser54 --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3. While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser54 completely suppressed the kinase activation. Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser54 --> Ala substitution could not be activated by the endogenous PKA in the intact cell. These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser54 in this domain plays a crucial role in activation.     

Identification of serines-1035/1037 in the kinase domain of the insulin receptor as protein kinase C alpha mediated phosphorylation sites

A new site of serine phosphorylation (Ser-1035/1037) has been identified in the kinase domain of the insulin receptor. Mutant receptors missing these two serines were expressed in Chinese hamster ovary cells overexpressing protein kinase C alpha. These mutant receptors lacked a phorbol ester-stimulated phosphoserine containing tryptic peptide as demonstrated by both high percentage polyacrylamide/urea gel electrophoresis and two-dimensional tlc. Moreover, a synthetic peptide with the sequence of this tryptic peptide was phosphorylated by isolated protein kinase C alpha and co-migrated with the phosphopeptide from in vivo labeled receptor. These results indicate that serine-1035 and/or 1037 in the kinase domain of the insulin receptor are phosphorylated in response to activation of protein kinase C alpha.     

The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1(ala252) allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Delta. The difference between the Pde1(ala252) allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2(val19) pde2Delta background, the Pde1(ala252) allele caused nearly the same hyperaccumulation of cAMP as pde1Delta, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1(ala252) mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1(ala252) enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1(ala252), and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.     

Protein kinase C-dependent regulation of Na+/Ca2+ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation

We compared the phosphorylation-dependent regulation of three mammalian Na+/Ca2+ exchanger isoforms (NCX1-NCX3) expressed in CCL39 fibroblasts that have little endogenous activity. Na+i-dependent 45Ca2+ uptake into NCX1- or NCX3-expressing cells, but not that into NCX2-expressing cells, was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB, which was abolished by pretreatment of cells with calphostin C or a prior long exposure to PMA. This suggests that NCX1 or NCX3, but not NCX2, is stimulated by a pathway involving protein kinase C (PKC). Immunoprecipitation experiments using [32P]orthophosphate-labeled cells revealed that both NCX2 and NCX3 proteins were phosphorylated to a much lesser extent than the NCX1 protein in unstimulated cells and that the extent of phosphorylation was not increased by treatment with PKC activators, although NCX1 phosphorylation was enhanced significantly. Using site-directed mutagenesis, we identified three phosphorylation sites in the NCX1 protein in the PMA-stimulated cells to be Ser-249, Ser-250, and Ser-357 with Ser-250 being predominantly phosphorylated. We found that the NCX1 mutant with these serine residues substituted with alanine still maintained a normal response to PMA. In contrast, the NCX1 or NCX3 mutant, with the large central cytoplasmic loop deleted, lost the responsiveness to PMA. These results suggest that the PKC-dependent regulation of NCX1 or NCX3 requires the central cytoplasmic loop but does not require the direct phosphorylation of the exchanger.     

Phosphorylation of the prostacyclin receptor during homologous desensitization. A critical role for protein kinase c

Agonist-induced phosphorylation of an epitope-tagged prostacyclin receptor (HAhIP) is mediated primarily by PKC (Smyth, E. M., Nestor, P. V., and FitzGerald G. A. (1996) J. Biol. Chem. 271, 33698-33704). Based on the two consensus sites for protein kinase C (PKC) phosphorylation in the C-terminal region mutant HAhIPs were generated: S328A and S374A, in which an alanine replaced Ser-328 or Ser-374, respectively, S328A/S374A and C-DEL, in which the C-terminal portion was truncated at amino acid 313. Mutant receptors, stably expressed in HEK293 cells, coupled normally to cAMP production. Substantially less coupling to inositol phosphate was apparent with S328A, S328A/S374A, and C-DEL compared with HAhIP or S374A. Point mutants resolved by SDS-polyacrylamide gel electrophoresis as a broad band with a molecular mass of 44-62, indicating that the receptors are glycosylated, and immunofluoresence staining demonstrated their membrane localization. C-DEL demonstrated a substantial reduction in glycosylation; bands with molecular masses of 38-54 (glycosylated), 30, and 27 kDa (unglycosylated) were apparent. Although membrane localization was evident, cellular localization was more diffuse. HAhIP and S374A underwent iloprost- and PMA-induced phosphorylation (1 and 5 microM, respectively, for 10 min). S328A and S328A/S374A showed a markedly less iloprost- and no PMA-induced phosphorylation. Phosphorylation of C-DEL was completely absent with either agonist. Electrospray mass spectrometry indicated that a peptide, including Ser-328, was phosphorylated in vitro by PKC, whereas one including Ser-374 was not. Iloprost (1 microM, 10 min) desensitized HAhIP- and S374A-mediated adenylyl cyclase activation. A less impressive desensitization was evident with S328A and S328A/S374A, and no desensitization of C-DEL coupling was apparent. Exposure of transfected cells to iloprost (1 microM) for increasing times induced a rapid desensitization of subsequent iloprost-induced (1 microM) HAhIP and S374A adenylyl cyclase coupling. In contrast, no significant time-dependent desensitization of S328A, S328A/S374A, or C-DEL coupling was evident. These results indicate that PKC-dependent phosphorylation is of critical importance to homologous regulation of hIP. Ser-328 is a primary site for PKC phosphorylation of hIP.     

Transforming growth factor-beta1 stimulates protein kinase A in mesangial cells

We recently demonstrated that transforming growth factor-beta (TGF-beta) stimulates phosphorylation of the type I inositol 1,4, 5-trisphosphate receptor (Sharma, K., Wang, L., Zhu, Y., Bokkala, S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine mesangial cells. In the present study, we evaluated whether TGF-beta stimulates PKA activation. Utilizing a specific PKA kinase assay, we found that TGF-beta increases PKA activity by 3-fold within 15 min of TGF-beta1 treatment, and the enhanced kinase activity was completely reversed by the inhibitory peptide for PKA (PKI; 1 microM). In mesangial cells transfected with a PKI expression vector, enhanced PKA activity could not be demonstrated with TGF-beta1 treatment. TGF-beta1 was also found to stimulate translocation of the alpha-catalytic subunit of PKA to the nucleus by Western analysis of nuclear protein as well as by confocal microscopy. TGF-beta1-mediated phosphorylation of cAMP response element-binding protein was completely reversed by H-89 (3 microM), a specific inhibitor of PKA. Stimulation of fibronectin mRNA by TGF-beta1 was also attenuated in cells overexpressing PKI. We thus conclude that TGF-beta stimulates the PKA signaling pathway in mesangial cells and that PKA activation contributes to TGF-beta stimulation of cAMP response element-binding protein phosphorylation and fibronectin expression.