B cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase Cdelta

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.     

The CREB leucine zipper regulates CREB phosphorylation, cardiomyopathy, and lethality in a transgenic model of heart failure

Signaling through cAMP plays an important role in heart failure. Phosphorylation of cAMP response element binding protein (CREB) at serine-133 regulates gene expression in the heart. We examined the functional significance of CREB-S133 phosphorylation by comparing transgenic models in which a phosphorylation resistant CREB-S133A mutant containing either an intact or a mutated leucine zipper domain (CREB-S133A-LZ) was expressed in the heart. In vitro, CREB-S133A retained the ability to interact with wild-type CREB, whereas CREB-S133A-LZ did not. In vivo, CREB-S133A and CREB-S133A-LZ were expressed at comparable levels in the heart; however, CREB-S133A markedly suppressed the phosphorylation of endogenous CREB, whereas CREB-S133A-LZ had no effect. The one-year survival of mice from two CREB-S133A-LZ transgenic lines was equivalent to nontransgenic littermate control mice (NTG), whereas transgenic CREB-S133A mice died with heart failure at a median 30 wk of age (P < 0.0001). CREB-S133A mice had an altered gene expression characteristic of the failing heart, whereas CREB-S133A-LZ mice did not. Left ventricular contractile function was substantially reduced in CREB-S133A mice versus NTG mice and only modestly reduced in CREB-S133A-LZ mice (P < 0.02). When considered in light of other studies, these findings indicate that overexpression of the CREB leucine zipper is required for both inhibition of endogenous CREB phosphorylation and cardiomyopathy in this murine model of heart failure.     

Phosphorylation of the cyclic AMP response element binding protein mediates transforming growth factor beta-induced downregulation of cyclin A in vascular smooth muscle cells

Transforming growth factor beta (TGFbeta), a multifunctional cytokine associated with vascular injury, is a potent inhibitor of cell proliferation. The current results demonstrate that the TGFbeta-induced growth arrest of vascular smooth muscle cells (VSMCs) is associated with cyclin A downregulation. TGFbeta represses the cyclin A gene through a cyclic AMP (cAMP) response element, which complexes with the cAMP response element binding protein (CREB). The CREB-cyclin A promoter interaction is hindered by TGFbeta, preceded by a TGFbeta receptor-dependent CREB phosphorylation. Induction of CREB phosphorylation with forskolin or 6bnz-cAMP mimics TGFbeta's inhibitory effect on cyclin A expression. Conversely, inhibition of CREB phosphorylation with a CREB mutant in which the phosphorylation site at serine 133 was changed to alanine (CREB-S133A) upregulated cyclin A gene expression. Furthermore, the CREB-S133A mutant abolished TGFbeta-induced CREB phosphorylation, cyclin A downregulation, and growth inhibition. Since we have previously shown that the novel PKC isoform protein kinase C delta (PKCdelta) is activated by TGFbeta in VSMCs, we tested the role of this kinase in CREB phosphorylation and cyclin A downregulation. Inhibition of PKCdelta by a dominant-negative mutant or by targeted gene deletion blocked TGFbeta-induced CREB phosphorylation and cyclin A downregulation. Taken together, our data indicate that phosphorylation of CREB stimulated by TGFbeta is a critical step leading to the inhibition of cyclin A expression and, thus, VSMC proliferation.     

Angiotensin II promotes the phosphorylation of cyclic AMP‐responsive element binding protein (CREB) at Ser133 through an ERK1/2-dependent mechanism

In cells from the adrenal medulla, angiotensin II (AII) regulates both the activity and mRNA levels of catecholamine biosynthetic enzymes whose expression is thought to be under the control of cAMP-responsive element (CRE) binding protein (CREB). In this study, we evaluated the effect of AII stimulation on CREB phosphorylation at Ser133 (pCREB) in bovine adrenal chromaffin cells (BACC). We found that AII produces a rapid and AII type-1 receptor (AT1)-dependent increase in pCREB levels, which is blocked by the MEK1/2 inhibitor U0126 but not by H-89, SB203580 or KN-93, suggesting that it is mediated by the extracellular-regulated protein kinases 1 and 2 (ERK1/2) and not by cAMP-dependent protein kinase (PKA), p38 mitogen-activated protein kinase (p38MAPK) or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) dependent pathways. Gel-shift experiments showed that the increase in pCREB levels is accompanied by an ERK1/2-dependent upregulation of CRE-binding activity. We also found that AII promotes a rapid and reversible increase in the activity of the non-receptor tyrosine kinase Src and that the inhibition of this enzyme completely blocks the AII-induced phosphorylation of ERK1/2, the CREB kinase (p90)RSK and CREB. Our data support the hypothesis that in BACC, AII upregulates CREB functionality through a mechanism that requires Src-mediated activation of ERK 1/2 and (p90)RSK.     

p90(RSK) blocks bad-mediated cell death via a protein kinase C-dependent pathway

Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser(112), a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser(112), suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90(RSK) and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser(112) both in vitro and in vivo. p90(RSK) stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser(112)-dependent manner. These findings suggest that p90(RSK) can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death.     

Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.     

UVA induces Ser381 phosphorylation of p90RSK/MAPKAP-K1 via ERK and JNK pathways

UVA exposure plays an important role in the etiology of skin cancer. The family of p90-kDa ribosomal S6 kinases (p90(RSK)/MAPKAP-K1) are activated via phosphorylation. In this study, results show that UVA-induced phosphorylation of p90(RSK) at Ser(381) through ERKs and JNKs, but not p38 kinase pathways. We provide evidence that UVA-induced p90(RSK) phosphorylation and kinase activity were time- and dose-dependent. Both PD98059 and a dominant negative mutant of ERK2 blocked ERKs and p90(RSK) Ser(381) phosphorylation, as well as p90(RSK) activity. A dominant negative mutant of p38 kinase blocked UVA-induced phosphorylation of p38 kinase, but had no effect on UVA-induced Ser(381) phosphorylation of p90(RSK) or kinase activity. UVA-induced p90(RSK) phosphorylation and kinase activity were markedly attenuated in JnK1(-/-) and JnK2(-/-) cells. A dominant negative mutant of JNK1 inhibited UVA-induced JNKs and p90(RSK) phosphorylation and kinase activity, but had no effect on ERKs phosphorylation. PD169316, a novel inhibitor of JNKs and p38 kinase, inhibited phosphorylation of p90(RSK), JNKs, and p38 kinase, but not ERKs. However, SB202190, a selective inhibitor of p38 kinase, had no effect on p90(RSK) or JNKs phosphorylation. Significantly, ERKs and JNKs, but not p38 kinase, immunoprecipitated with p90(RSK) when stimulated by UVA and p90(RSK) was a substrate for ERK2 and JNK2, but not p38 kinase. These data indicate clearly that p90(RSK) Ser(381) may be phosphorylated by activation of JNKs or ERKs, but not p38 kinase.     

(-)-Epigallocatechin gallate suppresses endothelin-1-induced interleukin-6 synthesis in osteoblasts: inhibition of p44/p42 MAP kinase activation

We previously showed that endothelin-1 (ET-1) stimulates the synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that protein kinase C (PKC)-dependent p44/p42 mitogen-activated protein (MAP) kinase plays a part in the IL-6 synthesis. In the present study, we investigated the effect of (-)-epigallocatechin gallate (EGCG), one of the major flavonoids containing in green tea, on ET-1-induced IL-6 synthesis in osteoblasts and the underlying mechanism. EGCG significantly reduced the synthesis of IL-6 stimulated by ET-1 in MC3T3-E1 cells as well primary cultured mouse osteoblasts. SB203580, a specific inhibitor of p38 MAP kinase, but not SP600125, a specific SAPK/JNK inhibitor, suppressed ET-1-stimulated IL-6 synthesis. ET-1-induced phosphorylation of p38 MAP kinase was not affected by EGCG. On the other hand, EGCG suppressed the phosphorylation of p44/p42 MAP kinase induced by ET-1. Both the IL-6 synthesis and the phosphorylation of p44/p42 MAP kinase stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of PKC, were markedly suppressed by EGCG. The phosphorylation of MEK1/2 and Raf-1 induced by ET-1 or TPA were also inhibited by EGCG. These results strongly suggest that EGCG inhibits ET-1-stimulated synthesis of IL-6 via suppression of p44/p42 MAP kinase pathway in osteoblasts, and the inhibitory effect is exerted at a point between PKC and Raf-1 in the ET-1 signaling cascade.     

Regulation of Protein Kinase A Activity by p90 Ribosomal S6 Kinase 1

Previously, we reported that the catalytic subunit of cAMP-dependent protein kinase (PKAc) binds to the active p90 ribosomal S6 kinase 1 (RSK1) (Chaturvedi, D., Poppleton, H. M., Stringfield, T., Barbier, A., and Patel, T. B. (2006) Mol. Cell. Biol. 26, 4586–4600). Herein, by overexpressing hemagglutinin-tagged RSK1 fragments in HeLa cells we have identified the region of RSK1 that is responsible for the interaction with PKAc. PKAc bound to the last 13 amino acids of RSK1, which overlaps the Erk1/2 docking site. This interaction between PKAc and RSK1 required the phosphorylation of Ser-732 in the C terminus of RSK1. Depending upon its phosphorylation status, RSK1 switched interactions between Erk1/2 and PKAc. In addition, a peptide corresponding to the last 13 amino acids of RSK1 with substitution of Ser-732 with Glu (peptide E), but not Ala (peptide A), decreased interactions between endogenous active RSK1 and PKAc. RSK1 attenuated the ability of cAMP to activate PKA in vitro and this modulation was abrogated by peptide E, but not by peptide A. Similarly, in intact cells, cAMP-mediated phosphorylation of Bcl-xL/Bcl-2-associated death promoter on Ser-115, the PKA site, was reduced when RSK1 was activated by epidermal growth factor, and this effect was blocked by peptide E, but not by peptide A. These findings demonstrate that interactions between endogenous RSK1 and PKAc in intact cells regulate the ability of cAMP to activate PKA and identify a novel mechanism by which PKA activity is regulated by the Erk1/2 pathway.     

Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.