Phosphorylation of glucocorticoid receptor tau1c transactivation domain enhances binding to CREB binding protein (CBP) TAZ2.

The glucocorticoid receptor (GR) N-terminal domain (NTD) contains a transactivation domain (activation function 1; AF-1). GR AF-1 is phosphorylated, but effects of this modification upon AF-1 activity and cofactor recruitment are not completely clear. GR AF-1 activity is mostly confined to a short unstructured domain called tau1c (amino acids 187-244) that contains three phosphorylation sites and binds a short cysteine rich fragment (CH3) of the coactivator CREB binding protein (CBP). Since the CH3 domain overlaps the CBP transcriptional adaptor zinc binding (TAZ) 2 domain, implicated in phosphorylation dependent binding to other unstructured transcription factor domains, we set out to investigate whether GR interacts with TAZ2 and whether this binding event is modulated by phosphorylation. We find that GR tau1c is absolutely required for enhancement of GR function and GR/CBP association in cultured cells. Tau1c interacts with TAZ2 in vitro and peptide mapping reveals CBP binding determinants throughout tau1c. Phosphorylation at GR Ser203, not involved in transactivation, does not affect tau1c/TAZ2 interactions. However, phosphorylation at Ser211 and Ser226, markers of GR transcriptional activity, greatly enhances TAZ2 binding in a synergistic fashion. We propose that GR tau1c phosphorylation could promote CBP recruitment and enhance AF-1 activity.

The day/night switch in KaiC, a central oscillator component of the circadian clock of cyanobacteria.

The circadian oscillator of the cyanobacterium Synechococcus elongatus is composed of only three proteins, KaiA, KaiB, and KaiC, which, together with ATP, can generate a self-sustained approximately 24 h oscillation of KaiC phosphorylation for several days. KaiA induces KaiC to autophosphorylate, whereas KaiB blocks the stimulation of KaiC by KaiA, which allows KaiC to autodephosphorylate. We propose and support a model in which the C-terminal loops of KaiC, the "A-loops", are the master switch that determines overall KaiC activity. When the A-loops are in their buried state, KaiC is an autophosphatase. When the A-loops are exposed, however, KaiC is an autokinase. A dynamic equilibrium likely exists between the buried and exposed states, which determines the steady-state level of phosphorylation of KaiC. The data suggest that KaiA stabilizes the exposed state of the A-loops through direct binding. We also show evidence that if KaiA cannot stabilize the exposed state, KaiC remains hypophosphorylated. We propose that KaiB inactivates KaiA by preventing it from stabilizing the exposed state of the A-loops. Thus, KaiA and KaiB likely act by shifting the dynamic equilibrium of the A-loops between exposed and buried states, which shifts the balance of autokinase and autophosphatase activities of KaiC. A-loop exposure likely moves the ATP closer to the sites of phosphorylation, and we show evidence in support of how this movement may be accomplished.

A microfluidic, high throughput protein crystal growth method for microgravity.

The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.