Phosphorylation of RelA/p65 on Serine 536 Defines an IκBα-independent NF-κB Pathway

Abstract

The association of the NF-B p65/p50 dimer with IB␣ plays a pivotal role in regulating its nuclear translocation and gene transcription. In addition, serine phosphorylation at various sites of the p65 subunit has been shown to be important in initiating transcription. Here we demonstrate that the regulation of nuclear translocation of p65 phosphorylated at serine 536 is not dependent on IB␣. Stimulation of either Jurkat or normal human T cells resulted in the nuclear translocation of phospho-p65 (Ser 536 ). In addition, the phospho-p65 (Ser 536 ) was not associated with either IB␣ or p50, and the nuclear translocation of phospho-p65 (Ser 536 ), but not total p65, was unaffected by the proteosome inhibitor MG-132, which blocks IB protein degradation and prevents p65/p50 dimer nuclear translocation. Accordingly, the co-expression of a dominant negative mutant of IB␣ blocked the transcriptional activity mediated by wild type but not the dominant positive p65 mutant (S536D). Furthermore, the transfection of the S536D form of p65 led to the induction of interleukin-8 transcription following stimulation, whereas the S536A form, which cannot be phosphorylated at this site, did not. Together, the findings suggest that p65 phosphorylated on serine 536 is not associated with or regulated by IB␣, that it has a distinct set of target genes, and that it may represent a noncanonical NF-B pathway that is independent of IB␣ regulation.The NF-B signaling pathway responds rapidly to a wide range of stimuli (1). Activation leads to the translocation of the transcription factors from the cytoplasm to the nucleus. The NF-B transcription factor consists of two subunits of either homo-or heterodimers of RelA/ p65, c-Rel, and p50. The complexes are held in the cytoplasm and prevented from activating transcription by a class of proteins referred to as inhibitors of NF-B or IB proteins. Upon stimulation, the IB proteins are phosphorylated by one of a number of IB kinases (IKK-␣, -␤, and -␥), ubiquitinylated, and degraded, which thereby allows the NF-B complex to translocate into the nucleus (2). However, recent findings have demonstrated the shuttling of the NF-B complex in and out of the nucleus in the absence of stimulation (3, 4). In addition to nuclear translocation of the NF-B complex, several studies have shown that the NF-B proteins are modified post-translationally, and those changes influence transcriptional activity. Examples of activation-induced posttranslational modifications include the acetylation of p65 to facilitate the retention of the NF-B complex in the nucleus (5, 6). In addition, the S-nitrosylation of cysteine 62 of p50 has been shown to affect the NF-B binding to DNA (7, 8).We have previously described the phosphorylation of various Rel proteins following the stimulation of T cells, and the phosphorylation of p50 increased the DNA binding capacity (9). Several studies have demonstrated the phosphorylation of p65 in response to various stimuli (10). Serine 276 of p65 is phosphorylated by protein kinase A during IB d...

Mutation of an IKK phosphorylation site within the transactivation domain of REL in two patients with B-cell lymphoma enhances REL's in vitro transforming activity

“…Namely, the S525P mutation affects REL's transactivating ability on the SOD2 and 3X-kB site promoters in different ways: REL-S525P induces decreased transactivation of the SOD2 promoter, but increased transactivation of the 3X-kB site promoter. Similar promoter context variability has also been seen when the IKK site Ser536 in RelA is mutated; that is, RelA-S536A transactivates the intercellular cell adhesion molecule (ICAM)-1 promoter more efficiently than wild-type RelA, but activates the IL-8 promoter weaker than RelA (Sasaki et al, 2005).…”

IL-1 receptor-associated kinase 1 is critical for latent membrane protein 1-induced p65/RelA serine 536 phosphorylation and NF-κB activation

“…Also, lymphotoxin ␤ induces NIK-and IKK␣-mediated p65 transcriptional activity through p65 S536 phosphorylation (29). Furthermore, phorbol 12-myristate 13-acetate and ionomycininduced p65 S536 phosphorylation is not inhibited by I B␣ expression, which is characteristic of the noncanonical pathway (49). Thus, the IRAK1 dependence of TES1-mediated NF-B activation is likely because of a need for p65 phosphorylation for efficient transcriptional enhancement by p65͞p52 complexes.…”

CD7/CD56-positive acute myeloid leukemias are characterized by constitutive phosphorylation of the NF-kB subunit p65 at Ser536

“…4 In the lymphoblastic lymphoma cell line Jurkat and in primary T-cell lymphocytes, p65 is constitutively phosphorylated at Ser536. 5 In this work we have isolated leukemic blasts obtained from two CD7/CD56-positive AML and seven conventional AML blasts. Both of the CD7/CD56 AML patients' samples were collected at the time of the diagnosis.…”

“…In a recently published work, it has been demonstrated that in Jurkat cells; and in primary T-cell lymphocyte, p65 is constitutively phosphorylated at Ser536. 5 Upon stimulation, phospho-p65 translocates into the nucleus where it mediates the transcription of a putative specific set of genes, probably different with respect to those activated by conventionally activated p65. [4][5] In Figure 1b, Jurkat cells extract has been considered as an example of known phosphorylated p65 expressing cells.…”

“…2 Recently, it has been demonstrated that NF-kB could be activated via a non-canonical pathway. [3][4][5] At least five kinases are responsible for the phosphorylation of p65 at Ser536. 4 This phosphorylated form of p65 is no longer subject to IkB-a inhibition and does not bind to p50.…”