Ser727-dependent transcriptional activation by association of p300 with STAT3 upon IL-6 stimulation

AbstractActivation of the signal transducer and activator of transcription 3 (STAT3) in response to interleukin-6 (IL-6) type cytokines involves both phosphorylation of Tyr705, which enables dimerization, nuclear translocation and DNA binding, as well as ser727 phosphorylation. Here, we describe that the 65 C-terminal amino acids of STAT3 can function as an independent transcription activation domain (TAD), particularly when a negative charge is introduced at position 727 by mutation of the serine residue into aspartate. The strong transcriptional activity of the C-terminal STAT3 Ser727Asp TAD is coupled to a constitutive association with the co-activator p300. In HepG2 cells, p300 associates with STAT3 upon IL-6 stimulation, and overexpression of p300 enhances the transcriptional activity of STAT3α, but not of STAT3β or STAT3 Ser727Ala. We conclude that Ser727 phosphorylation in the C-terminal region of STAT3 is required for transactivation by association with p300

Whole Exome Sequencing in Multi-Incident Families Identifies Novel Candidate Genes for Multiple Sclerosis

Multiple sclerosis (MS) is a degenerative disease of the central nervous system in which auto-immunity-induced demyelination occurs. MS is thought to be caused by a complex interplay of environmental and genetic risk factors. While most genetic studies have focused on identifying common genetic variants for MS through genome-wide association studies, the objective of the present study was to identify rare genetic variants contributing to MS susceptibility. We used whole exome sequencing (WES) followed by co-segregation analyses in nine multi-incident families with two to four affected individuals. WES was performed in 31 family members with and without MS. After applying a suite of selection criteria, co-segregation analyses for a number of rare variants selected from the WES results were performed, adding 24 family members. This approach resulted in 12 exonic rare variants that showed acceptable co-segregation with MS within the nine families, implicating the genes MBP, PLK1, MECP2, MTMR7, TOX3, CPT1A, SORCS1, TRIM66, ITPR3, TTC28, CACNA1F, and PRAM1. Of these, three genes (MBP, MECP2, and CPT1A) have been previously reported as carrying MS-related rare variants. Six additional genes (MTMR7, TOX3, SORCS1, ITPR3, TTC28, and PRAM1) have also been implicated in MS through common genetic variants. The proteins encoded by all twelve genes containing rare variants interact in a molecular framework that points to biological processes involved in (de-/re-)myelination and auto-immunity. Our approach provides clues to possible molecular mechanisms underlying MS that should be studied further in cellular and/or animal models

Proteolytic degradation of Smad4 in extracts of AML blasts

Loss of transforming growth factor (TGF) β signaling has been implicated in malignant transformation of various tissues. To investigate a potential role of Smad4 in acute myeloid leukemia (AML), the expression of Smad4 was determined in blast cells from AML patients. Western analysis of nuclear extracts of nine AML samples indicated the absence of Smad4 protein in two cases. Smad4 RT-PCR analysis of these cases indicated normal Smad4 mRNA expression, and sequencing of one of these cases revealed no mutations as compared to wild type Smad4. Next, it was investigated whether Smad4 protein from these AML cases was subject to proteolytic degradation by incubating cell extracts of these Smad4-negative AML cells with extracts from COS-7 cells in which a tagged Smad4 was overexpressed. Inhibitor studies indicated that the extracts of AML blasts lacking Smad4 possessed a serine-dependent proteolytic activity, capable of degrading Smad4. Transfection studies using an SBE containing reporter construct as well as RT-PCR analysis of endogenous TGFβ1 responsive genes indicated that the AML blasts were still able to respond to TGFβ1, despite the observed degradation of Smad4. It was, therefore, concluded that the degradation of Smad4 was possibly AML subtype-dependent, in vitro phenomenon, occurring during the preparation of nuclear and cellular extracts despite the addition of a protease inhibitor cocktail. The results indicate that care should be taken when interpreting data obtained from protein expression studies using AML blast cells.

LIF-Induced STAT3 Signaling in Murine versus Human Embryonal Carcinoma (EC) Cells

Self-renewal and the maintenance of pluripotency of mouse embryonal stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF). Mouse ES cells can be cultured and kept undifferentiated in the absence of embryonal feeder-cell layers when exogenous LIF concentrations are maintained above a threshold concentration. An important downstream target of LIF signal transduction in mouse ES cells is the transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast to mouse ES cells, human ES cells are unresponsive to LIF and depend on feeder cells for undifferentiated growth. Here, we investigated the activation patterns of LIF-downstream effectors in mouse and human embryonal carcinoma (EC) cells. We report that LIF induces both ERK-1 as well as STAT3 activation in mouse P19 EC cells. LIF enhances the proliferation rate of P19 EC cells, which depends on ERK activity but does not require activation of STAT3. In contrast, LIF does not activate STAT3, ERK, or the gp130 receptor in human N tera-2/D1 EC cells, although all receptor components are expressed. The negative feedback protein suppressor of cytokine signaling 1 (SOCS-1) is constitutively expressed in N tera-2/D1 EC cells, suggesting that LIF signal transduction is inhibited by elevated levels of SOCS-1 expression.

Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line - Pathways that are shared with and distinct from IL-10

IL (interleukin)-22 is an IL-10-related cytokine; its main biological activity known thus far is the induction of acute phase reactants in liver and pancreas. IL-22 signals through a receptor that is composed of two chains from the class II cytokine receptor family: IL-22R (also called ZcytoR11/CRF2-9) and IL-10Rbeta (CRF2-4), which is also involved in IL-10 signaling. In this report, we analyzed the signal transduction pathways activated in response to IL-22 in a rat hepatoma cell line, H4IIE. We found that IL-22 induces activation of JAK1 and Tyk2 but not JAK2, as well as phosphorylation of STAT1, STAT3, and STAT5 on tyrosine residues, extending the similarities between IL-22 and IL-10. However our results unraveled some differences between IL-22 and IL-10 signaling. Using antibodies specific for the phosphorylated form of MEK1/2, ERK1/2, p90RSK, JNK, and p38 kinase, we showed that IL-22 activates the three major MAPK,PK pathways. IL-22 also induced serine phosphorylation of STAT3 on Ser(727). This effect, which is not shared with IL-10, was only marginally affected by MEK1/2 inhibitors, indicating that other pathways might be involved. Finally, by overexpressing a STAT3 S727A mutant, we showed that serine phosphorylation is required to achieve maximum transactivation of a STAT responsive promoter upon IL-22 stimulation