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Regulation of Smad signaling by protein kinase C¹

IHOR YAKYMOVYCH^*,, PETER TEN DIJKE^*,, CARL-HENRIK HELDIN^* and SERHIY SOUCHELNYTSKYI^*2

^* Ludwig Institute for Cancer Research, Box 595, Uppsala, Sweden;
Institute of Biochemistry of National Academy of Sciences of Ukraine, Lviv, Ukraine;
Netherlands Cancer Institute, 1066CX, Amsterdam, Netherlands

²Correspondence: Ludwig Institute for Cancer Research, Box 595, Husargatan (str), 3, Biomedical Center, S-751 24, Uppsala, Sweden. E-mail: serhiy.souchelnytskyi{at}licr.uu.se

SPECIFIC AIMS

We investigate a mechanism through which Smad-dependent transforming growth factor beta (TGF-ß) signaling is modulated by protein kinase C (PKC). PKC directly phosphorylates receptor-regulated Smad proteins. It also abrogates the ability of Smad3 to bind directly to DNA, which leads to subsequent impairment of transcriptional responses dependent on the direct binding of Smad3 to DNA and to down-regulation of the growth inhibitory and pro-apoptotic action of TGF-ß.

PRINCIPAL FINDINGS

1. Receptor-regulated Smads are directly phosphorylated by PKC
We found that treating cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in appearance of additional tryptic phosphopeptides in 2-dimensional maps of endogenous Smad3 and Smad2 from Mv1Lu cells, and in maps of Smad3, Smad2, and Smad1 overexpressed in Mv1Lu or COS-1 cells. The PKC-dependent phosphorylation was observed after only 5 min of treatment with PMA, and maximal effect was observed after 60 min. The appearance of these phosphopeptides was inhibited when cells were pretreated with the PKC inhibitors calphostin C or staurosporine, which act by blocking regulatory and catalytic subunits of PKC, respectively. No effect of the MEK-1 inhibitor, PD98059, on PMA-induced phosphorylation was observed. The PKC-induced phosphopeptides were also detected when purified Smad3 or Smad2 were phosphorylated directly in vitro by purified catalytic subunit of PKC. These data show that Smad3 and Smad2 are substrates of PMA-activated PKC.

Phosphoamino acid analysis and Edman degradation of ³²P-labeled tryptic peptides suggested that Ser37 and Ser70 in Smad3 and Ser47 and Ser110 in Smad2 were phosphorylated by PKC. Phosphopeptide mapping of Smad2 and Smad3, with the respective serines mutated to alanine residues, confirmed that Ser37 and Ser70 in Smad3 and Ser47 and Ser110 in Smad2 are phosphorylation sites for activated PKC.

2. Phospholipase C (PLC), but not phosphatidylinositol 3-kinase (PI3K) or MAP kinase, is involved in PKC-dependent phosphorylation of Smads
Stimulation of Mv1Lu cells with fetal bovine serum led to appearance of PKC-dependent phosphopeptides in maps derived from Smad3. Pretreatment of cells with the PLC inhibitor U73122 or with the PKC inhibitor calphostin C led to a decrease of the PKC-dependent phosphopeptides induced by serum, whereas inhibitors of phosphatidylinositol 3-kinase (LY294002) and MEK-1 (PD98059) did not block their appearance. Thus, the mitogen-induced activation of PLC followed by PKC activation leads to phosphorylation of the MH1 domain of Smad3.

3. PKC-dependent phosphorylation abrogates direct DNA binding of Smad3
Based on the 3-dimensional structure of the MH1 domain of Smad3, both the PKC phosphorylatable serines are located on the surface that is facing DNA and phosphorylation of these sites, with introduction of negatively charged phosphogroups, which may affect the interaction with DNA (Fig. 1A ). To investigate this possibility, we prepared GST-fusion proteins of wild-type and serine mutants of Smad3. Constructs with deleted MH2 domains were used to unravel the DNA binding properties of the MH1 domain. We found that the introduction of negatively charged aspartic acid residues instead of Ser37 or Ser70 in GST-Smad3MH1 abrogated the DNA binding (Fig. 1B ). In accordance with this observation, the phosphorylation of purified wild-type GST-Smad3MH1 by the catalytic subunit of PKC strongly inhibited the DNA binding (Fig. 1C ).

View larger version (72K): [in this window] [in a new window]   Figure 1. PKC phosphorylation sites are involved in regulation of direct DNA binding by Smad3. A) Location of PKC phosphorylation sites in the 3D structure of the MH1 domain of Smad3 (modified from Shi et al. (1998) Cell 94, 585). B) Replacement of Ser37 or Ser70 for aspartic acid residues, abrogates the direct DNA binding of GST-Smad3MH1 (GST-Smad3). Wild-type and serine mutants of GST-Smad3MH1 were incubated with 32P-labeled ‘4xWT’ probe containing Smad binding elements, and electrophoretic-mobility shift assay was performed. C) Phosphorylation in vitro of the GST-Smad3MH1 (Smad3) by a purified catalytic subunit of PKC leads to decrease in DNA binding. D) Mutations of Ser37 and Ser70 in Smad3 abrogate the DNA binding by ectopically expressed full-length Smad3. Electrophoretic-mobility shift assay was performed with nuclear extracts of Mv1Lu cells stably transfected with full-length wild-type or mutated myc-Smad3. All cells were treated with TGFß1 (10 ng/ml) and where indicated with PMA (0.1 µM) for 1.5 h, and lysates were collected. For the supershift, anti-myc antibodies were used.

When DNA binding was evaluated by using nuclear extracts of cells stably transfected with wild-type myc-Smad3, the treatment of cells with PMA resulted in a decrease of formation of the specific myc-Smad3 containing complex (Fig. 1D ). The presence of myc-Smad3 in a DNA-binding complex was evaluated by the ability of anti-myc antibodies to shift the complex.

4. PKC phosphorylation affects transcriptional activity of Smad3, but not of Smad2
The effect of PMA treatment and mutation of Smad3 on the activation of the (CAGA)₁₂-Luc reporter, which contains a multimerized Smad binding element, was then investigated. PMA treatment of cells inhibited the TGF-ß1-induced response in mock-transfected cells. We also observed an inhibition of ligand-dependent stimulation of luciferase reporters in cells overexpressing S37D and S70D mutants of Smad3. Moreover, the TGF-ß-dependent stimulation of cells overexpressing the S70A mutant was weaker than for cells transfected with the wild-type, which suggests that the S70A mutant in vivo is less potent than wild-type Smad3 in mediating signaling. Therefore, the effects on the activation of (CAGA)₁₂-Luc reporter correlated with the DNA binding abilities of Smad3 full-length mutants. Similar results were obtained by using other reporters containing the Smad binding element, SBE₄-Luc and p800-luc, or by analysis of junB mRNA expression by Northern blotting. It supports the conclusion that the PKC-dependent phosphorylation has a negative effect on the Smad3-mediated transcriptional regulation.

Smad2 and Smad3 are activated by the same ligands, TGF-ß and activin, but their modes of action differ. Smad2, in contrast to Smad3, cannot bind DNA directly. We found that the Smad2 mutants with the PKC phosphorylatable serine residues changed to alanine or that aspartic acid activated the Smad2-responsive ARE-Luc reporter equally efficiently as wild-type Smad2. For activation of the activin-responsive element containing ARE-Luc reporter, Smad2 does not bind DNA directly. Therefore, whereas PKC-dependent phosphorylation in the MH1 domain abrogates direct DNA binding activity, it does not interfere with transactivation of target genes, which can be mediated by the MH2 domain.

5. Interference with PKC-dependent phosphorylation of Smad3 leads to increased PMA-dependent foci formation and is important for PMA inhibition of TGF-ß-induced cell death.
We found that cells stably transfected with the DNA binding-impaired S37D, S70A, and S70D mutants of Smad3 upon simultaneous stimulation with PMA and TGF-ß1, formed transformation foci. Cells stimulated with TGF-ß1 or PMA individually did not form foci under the same conditions. Non-transfected cells and cells transfected with wild-type Smad3 or the S37A mutant began to overgrow in response to stimulation with TGF-ß1 and PMA compared with non-transfected cells but did not form foci. This condition is probably due to noncomplete inactivation by endogenous PKC of the intracellular pool of Smad3, which still allows formation of some residual DNA binding complex (Fig. 1D ). Thus, the perturbation of Smad3 binding to DNA enhances the transforming effect of PMA and TGF-ß1.

Previously we showed that TGF-ß induces apoptosis of Mv1Lu cells upon culturing in serum-deprived medium. However, simultaneous treatment of cells with PMA abrogated this effect. Similar effects were observed in Mv1Lu cells stably transfected with the wild-type myc-Smad3. As expected, stable transfection of cells, with the DNA binding-impaired Smad3 mutants with the PKC phosphorylation sites replaced with aspartic acid residues, abrogated the TGF-ß-dependent induction of cell death even in the absence of PMA.

CONCLUSIONS

Inactivation of TGF-ß-induced growth inhibition and apoptosis are important steps in cell transformation during tumorigenesis. Smad inactivation has been found in various human cancers; thus Smads are considered tumor suppressors. Smad inactivation can be achieved by deletions or point mutations of their genes, but also through functional inactivation via accelerated degradation or via transcriptional repression, or through MAP kinase-dependent phosphorylation of Smads, which leads to abrogation of their nuclear translocation. We show here another phosphorylation-dependent mechanism involving PKC, which selectively down-regulates certain TGF-ß signals (Fig. 2 ).

View larger version (138K): [in this window] [in a new window]   Figure 2. Model of regulatory cross talk between TGF-ß Smad-dependent signaling and PKC. The phosphorylation of TGFß receptor-activated Smads by PKC results in abrogation of direct DNA binding of Smad3 but does not affect transactivation function, which is mediated by interaction with transcription factors as demonstrated for Smad2 with FAST-1. PKC-mediated phosphorylation can occur not only subsequent to receptor phosphorylation (as shown), but also prior to this event. TyrKR, tyrosine kinase receptors; GPCR, G-protein coupled receptors.

The two identified PKC phosphorylation sites are located at a distance of 33 amino acid residues in Smad3; however, in the folded 3-dimensional structure they are both located on the same surface of the MH1 domain. This surface is facing DNA, thus it is not surprising that the DNA binding function of Smad3 is affected (Fig. 1) . Introduction of a negative charge by phosphorylation, or by mutagenesis at these sites, abrogated the direct DNA binding of Smad3 with subsequent inhibition of transcriptional responses.

Stimulation of PKC activity by TGF-ß has been reported. Thus, it is possible that TGF-ß initiates a negative feedback mechanism by activating PKC, which, via phosphorylation of Smad3, inhibits its function as DNA-binding transcription factor.

As a potent inhibitor of cell growth, compromise of TGF-ß in its signaling upon cell transformation is expected. Loss of DNA binding ability of Smad3 makes cells more sensitive to transformation by PMA and TGF-ß, measured as foci formation, and inhibits cell death induced by TGF-ß. Therefore, our data implicate Smad3 as a point of cross talk for the TGF-ß inhibitory and PKC-mediated stimulatory effects on cell growth (Fig. 2) .

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0474fje ; to cite this article, use FASEB J. (January 5, 2001) 10.1096/fj.00-0474fje

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This Article Full Text (PDF) All Versions of this Article: 15/3/553 00-0474fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by YAKYMOVYCH, I. Articles by SOUCHELNYTSKYI, S. Search for Related Content PubMed PubMed Citation Articles by YAKYMOVYCH, I. Articles by SOUCHELNYTSKYI, S.