Packing, specificity, and mutability at the binding interface between the p160 coactivator and CREB-binding protein

Among the most common interaction motifs between nuclear proteins is the recognition of one or more amphipathic helices. In an effort to determine principles behind this recognition, we have investigated the interaction between the p160 coactivator protein ACTR and the ACTR-binding domain of the CREB-binding protein, CBP. The two proteins use relatively small portions of their primary sequences to form a single synergistically folded domain consisting of six intertwined alpha-helices, three from each protein. Neither of the component polypeptides forms a cooperatively folded domain in isolation. However, a considerable amount of residual secondary structure remains in the isolated CBP domain according to CD spectroscopy. Chemical denaturation, differential scanning calorimetry, and ANS binding experiments demonstrate that the isolated CBP domain is not entirely unfolded but forms a helical state with the characteristics of a molten globule. Mutations probing the functional and energetic significance of a buried intermolecular Arg-Asp salt bridge in the interface of the protein complex suggest that these residues are tuned for functional discrimination and not strictly for binding affinity or stability. These results suggest a mechanism for formation of the complex where the unfolded ACTR domain interacts with the partly folded CBP domain in a rapid and specific manner to form the final stable complex.     

Magnolol induces the distributional changes of p160 and adipose differentiation-related protein in adrenal cells

Magnolol stimulates adrenal steroidogenesis and induces the distributional changes of p160 and adipose differentiation-related protein (ADRP) in rat adrenal cells. This study investigated the underlying signaling mechanisms involved in these processes. Magnolol (30 M) caused a time-dependent increase in the phosphorylation of extracellular signal-related kinase (ERK) in cultured adrenal cells. The following evidence supports a link between ERK activation and p160 translocation. First, the magnolol-induced redistribution of p160 from the lipid droplet surface to the cytosol, resulting in the decrease in the percentages of p160-positive cells, and this decrease in p160-positive cells was completely blocked by pretreatment with either of the MAPK-ERK kinase (MEK) inhibitors PD98059 or U0126. Second, magnolol did not significantly decrease total p160 protein levels but caused an increase in threonine phosphorylation of p160, which reached a maximum after 5 min of magnolol treatment, and this magnolol-induced phosphorylation of p160 was prevented by pretreatment with U0126, suggesting the involvement of ERK. In addition, magnolol decreased both ADRP immunostaining intensity at the lipid droplet surface and the percentage of ADRP-positive cells. This was further confirmed biochemically by the decrease in ADRP levels in total cell homogenates and in lipid droplet fractions. Magnolol-induced decrease in ADRP staining at the lipid droplet surface was not affected by pretreatment with PD98059 or U0126, indicating that ERK signaling was not involved in this event. Furthermore, treatment with 30 M magnolol for 6 h resulted in about 50% decrease in ADRP protein level. Therefore, decreased protein levels of p160 and ADRP at the lipid droplet surface induced by magnolol were mediated via two different mechanisms: phosphorylation of p160 and downregulation of ADRP expression, respectively.     

Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP-1

We recently characterized the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 were sufficient to confer binding to PARP-1, whereas the amino-terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP-1 and overexpression of PARP-1. Our results unequivocally show that the presence of PARP-1 regulates the basal expression of wt p53 in unstressed cells.