Murine Protein Serine/Threonine Kinase 38 Stimulates TGF-β Signaling in a Kinase-dependent Manner via Direct Phosphorylation of Smad Proteins

Abstract

The present study demonstrated that murine protein serine/ threonine kinase 38 (MPK38) coimmunoprecipitates with Smad proteins (Smad2, -3, -4, and -7) and that this association is mediated by the catalytic kinase domain of MPK38. The association between MPK38 and Smad2, -3, and -4 was significantly increased by TGF-␤ or ASK1 signals, whereas these signals decreased association of MPK38 with Smad7. MPK38 stimulated TGF-␤-induced transcription required for TGF-␤-mediated biological functions, such as apoptosis and cell growth arrest, in a kinase-dependent manner. Knockdown of endogenous MPK38 showed an opposite effect, inhibiting TGF-␤ signaling. MPK38-mediated phosphorylation of Smad proteins (Ser 245 of Smad2, Ser 204 of Smad3, Ser 343 of Smad4, and Thr 96 of Smad7) was also found to be crucial to the positive regulation of TGF-␤ signaling induced by MPK38. In addition, MPK38 enhanced nuclear translocation of Smad3, as well as redistribution of Smad7 from the nucleus to the cytoplasm, in response to TGF-␤. Together, these results indicate that MPK38 functions as a stimulator of TGF-␤ signaling through direct interaction with and phosphorylation of Smad proteins.Type I transforming growth factor-␤ (TGF-␤) receptor kinases are serine/threonine kinases with roles in a wide array of cellular processes. Phosphorylation of the C-terminal SXS motif in receptor-regulated Smads (R-Smads) by these kinases is a crucial step in TGF-␤ family signaling (1-3). R-Smad phosphorylation results in the formation of heterodimeric complexes with the common Smad, Smad4. In addition, the phosphorylation of serine or threonine residues in the N-terminal or linker region has been observed in endogenous R-Smads (4). Several lines of evidence have demonstrated the existence of kinases responsible for N-terminal or linker region phosphorylation events, including mitogen-activated protein kinases (MAPKs), Ca 2ϩ and calmodulin-dependent kinase II, cyclindependent kinase (CDK), 3 protein kinase C (PKC), and G protein-coupled receptor kinase 2 (5). Common Smad, Smad4, is constitutively phosphorylated in cells, although not all of the phosphorylation sites are known. Extracellular signal-regulated kinase (ERK) has been shown to phosphorylate Smad4 at Thr 277 , resulting in the prevention of nuclear translocation of Smad4 (6). The inhibitory Smads (I-Smads), Smad6 and Smad7, are phosphorylated by uncharacterized kinases (7,8). This provides a mechanism for integration of the Smad pathway with other signaling pathways, in which kinases act as essential mediators, which modulate Smad-mediated signaling. Thus, the identification and characterization of additional cellular kinases responsible for Smad phosphorylation could lead to a better understanding of the regulatory role of Smad phosphorylations, especially in the N-terminal and linker region, in TGF-␤ signaling.Murine protein serine/threonine kinase 38 (MPK38), also known as pEg3 kinase and maternal embryonic leucine zipper kinase (Melk), is a member of the AMP-activated protein kinase family o...

Murine Protein Serine-threonine Kinase 38 Activates p53 Function through Ser15 Phosphorylation

“…The Mpk38-specific siRNA oligonucleotide has also been described previously (20). The p53 substitution mutants used for the in vitro kinase assays were generated by PCR using wild-type p53 as the template, as described previously (20,21). The following mutant and wild-type p53 primers were used: p53(S15A) forward, 5Ј-GCCCCCTCTGGCTCAG-GAAACAT-3Ј, and reverse, 5Ј-ATGTTTCCTGAGCCAGAG-GGGGC-3Ј; p53(T118A) forward, 5Ј-CATTCTGGGGCGGC-CAAGTCT-3Ј, and reverse, 5Ј-AGACTTGGCCGCCCCAGA-ATG-3Ј; p53(S260A) forward, 5Ј-CTGGAAGACGCGAGTG-GTAAT-3Ј, and reverse, 5Ј-ATTACCACTCGCGTCTTCCAG-3Ј; and wild-type p53 forward, 5Ј-GCGAATTCATGGAGGAGC-CGCAGTCA-3Ј containing an EcoRI site (underlined), and reverse, 5Ј-GCCTCGAGTCAGTCTGAGTCAGGCCC-3Ј containing an XhoI site (underlined).…”

“…MPK38 Kinase Assay-In vitro kinase assays using recombinant MPK38 proteins were performed as described previously (21). For in vitro kinase assays using MPK38 proteins purified from HEK293 cells expressing GST-MPK38, cells were transiently transfected with pEBG-Mpk38 (a GST tagged MPK38-encoding expression vector) using WelFect-Ex TM Plus and solubilized with lysis buffer (20 mM HEPES, pH 7.9, 10 mM EDTA, 0.1 M KCl, and 0.3 M NaCl).…”

“…The cleared lysates were then precipitated by glutathione-Sepharose beads. The GST precipitates were washed three times with lysis buffer and twice with kinase buffer (50 mM HEPES, pH 7.4, 1 mM DTT, and 10 mM MgCl 2 ) and were then subjected to an in vitro kinase assay, as described previously (20,21), using recombinant p53 as a substrate in the presence of 5 Ci of [␥-32 P]ATP, followed by SDS-PAGE and autoradiography.…”

“…WB, Western blot; TAD, transactivation domain; OD, oligomerization domain; BD, basic domain. AMPK consensus motif ⌽-(X, basic)-XX(S/T)XXX-⌽, where ⌽ is a hydrophobic residue (21,22 4A, right panel), indicating that the Ser 15 within the aminoterminal region of p53 represents a potential phosphorylation site for MPK38. This was also confirmed by immunoblot analysis using an anti-phospho-p53(S15) antibody (Fig.…”

“…B, modulation of p53 target genes by overexpression and knockdown of MPK38. The following cells were used for immunoblot analyses: parental HCT116 cells; HCT116 cells stably overexpressing MPK38 (MPK38(OE)); HCT116 cells stably expressing Mpk38-specific shRNA (MPK38(KD)); MPK38(KD) cells (MPK38(KD)ϩW) transfected with ϳ3 g of GST-Mpk38 wobble mutant (20); HCT116 cells transfected with 200 nM of control scrambled siRNA (Sc) or Mpk38-specific siRNA (MPK38); and inducible Mpk38 shRNA HEK293 cells (21). The cells were lysed and subjected to immunoblot analysis using anti-p53, anti-phospho-p53(S15), anti-p21, anti-MDM2, anti-BAX, anti-MPK38, anti-␤-actin, and anti-GST antibodies.…”