Abstract
The Aristaless-related homeobox protein (ARX) is a transcription factor expressed in the developing forebrain, skeletal muscle, pancreas, testis, and a variety of other tissues. It is known to have context-dependent transcriptional activator and repressor activity, although how it can achieve these opposing functions remains poorly understood. We hypothesized phosphorylation status might play a role in pivoting ARX between functioning as an activator or repressor. To gain further mechanistic insight as to how ARX functions, we identified multiple phosphorylation sites on ARX. We further established PKA as the kinase that phosphorylates ARX at least at Ser266 in mice. Two other kinases, CK2α and CDK4/cyclin D1, were also identified as kinases that phosphorylate ARX in vitro. Unexpectedly, phosphorylation status did not change either the nuclear localization or transcriptional function of ARX.
Highlights
Post-translational modification of proteins is an essential cellular process that influences the protein localization, function, and degradation
Both calf intestinal alkaline phosphatase (CIAP) and Calyculin A (CA) treatments resulted in electrophoretic mobility shifts for the 1–90 and 90–392 fragments
These data suggest that Aristaless-related homeobox protein (ARX) phosphorylation sites reside primarily in the N terminal two-thirds of ARX, or the phosphorylation of the C-terminal is highly sensitive to the 3-dimensional structure that is disrupted when truncated
Summary
Post-translational modification of proteins is an essential cellular process that influences the protein localization, function, and degradation. Phosphorylation is one of the most common modifications that can take place at one or multiple sites, and at different rates along the protein to modulate their functions [1,2,3]. The activity of transcription factors could be regulated by phosphorylation and many transcriptional factors have been shown to be phosphorylated at multiple sites by one or multiple kinases [4,5]. Sp1, a well-known bi-functional (activator and inhibitor) transcription factor, can be phosphorylated by ERK [6], PKC [7], CK2 [8], CDK1 [9], as well as many other kinases. In smooth muscle cells, Sp1 functions as a transcriptional activator after phosphorylation by ERK, but is switched to a repressor of platelet-derived growth factor receptor α (PDGFR-α) transcription after phosphorylation triggered by fibroblast growth factor 2 (FGF-2) [11]
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