5 th Discussions in Struc tural Molecular Bi ol o gy Conference Committee Lec tures-Thursday, March 16 L1 REGULATION BY MOLECULAR INTERFERENCE: HOW 14-3-3 PROTEIN CONTROLS ACTIVITY OF FORKHEAD TRANSCRIPTION FACTOR FOXO4
Trends in Bio chem i-cal Sci ences
The 14-3-3 pro teins, a fam ily of dimeric reg u la tory proteins , are in volved in many bi o log i cally im por tant processes. The com mon fea ture of 14-3-3 pro teins is their abil ity to bind to other pro teins in a phosphorylation-depend ent man ner. Through these bind ing in ter ac tions, 14-3-3 pro teins work as mo lec u lar scaf folds, mod u lat ing the bi o log i cal func tions of their part ners. 14-3-3 pro teins rec og nize short mo tifs con tain ing a phosphorylated serine or
... ine res i due. FOXO tran scrip tion fac tors are prom-is ing can di dates to serve as mo lec u lar links be tween lon-gev ity and tu mor sup pres sion. These fac tors are ma jor sub strates of the pro tein kinase B (Akt). In the pres ence of in su lin and growth fac tors, FOXO pro teins are relocalized from the nu cleus to the cy to plasm and de graded via the ubiquitin-proteasome path way. In the ab sence of growth fac tors, FOXO pro teins translocate to the nu cleus and upregulate a se ries of tar get genes, thereby pro mot ing cell cy cle ar rest, stress re sis tance, or apoptosis [1,2]. Un der con di tions of low pro tein kinase B (PKB) ac tiv-ity (in the ab sence of growth fac tors), FOXO pro teins are pre dom i nantly nu clear and the rate of im port ex ceeds the rate of ex port. This shift in equi lib rium prob a bly oc curs because the bind ing of FOXO to DNA an chors FOXO within the nu cleus. Fol low ing the ac ti va tion of PKB (PKB-reg u-lated shut tling) by in su lin-phosphatidylinositol-3-kinase (PI3K) sig nal ling, PKB translocates to the nu cleus. A 14-3-3 pro tein de void of ligand en ters the nu cleus passively. PKB phos phory lates FOXO, prob a bly on mul ti ple sites, and PKB-me di ated phosphorylation in duces the bind ing of 14-3-3 pro tein to the FOXO pro teins in the nucleus , which re sults in the re lease of the FOXO pro tein from the DNA. Fol low ing translocation to the cytosol, the bound 14-3-3 pro tein pre vents re-en try into the nu cleus by mask ing the NLS and in hib it ing importin bind ing. This results in an equi lib rium shift to wards the cytosol. Main goal of our re search was to elu ci date the mo lec u-lar mech a nism of 14-3-3 pro tein-de pend ent reg u la tion of FOXO4 func tion. We have shown that phosphorylation of FOXO4 by pro tein kinase B at Thr-28 and Ser-193 cre ates two 14-3-3 bind ing mo tifs. An a lyt i cal gel fil tra tion and sed i men ta tion equi lib rium ex per i ments in di cate that doubly phosphorylated FOXO4 and 14-3-3zeta form a complex with 1:2 mo lar stoichiometry and a K(D) of less than 30 nM. An ac tive role for 14-3-3 in the dis as sem bly of the FOXO4/DNA com plex is dem on strated by the fact that, in the pres ence of 14-3-3, two phosphorylated 14-3-3 bind ing mo tifs are needed for the com plete in hi bi tion of FOXO4 bind ing to its tar get DNA . We have also in ves ti gated whether the phosphorylation by pro tein kinase B, the 14-3-3 pro tein, and DNA bind ing af fect the struc ture of FOXO4 nu clear lo cal iza tion se quence (NLS). We have used site-di rected la bel ing of FOXO4 NLS with the ex trin-sic fluorophore 1,5-IAEDANS in con junc tion with steady-state and time-re solved flu o res cence spec tros copy to study conformational changes of FOXO4 NLS in vi tro. Our data show that the 14-3-3 pro tein bind ing sig nif i cantly changes the en vi ron ment around AEDANS-la beled NLS and re duces its flex i bil ity. On the other hand, the phosphorylation it self and the bind ing of dou ble-stranded DNA have a small ef fect on the struc ture of this re gion . 1. B.M.T. Burgering and G.