Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX.

AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. We show that AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity.

Presenilin 2 interacts with sorcin, a modulator of the ryanodine receptor.

Perturbed Ca(2+) homeostasis is a common molecular consequence of familial Alzheimer's disease-linked presenilin mutations. We report here the molecular interaction of the large hydrophilic loop region of presenilin 2 (PS2) with sorcin, a penta-EF-hand Ca(2+)-binding protein that serves as a modulator of the ryanodine receptor intracellular Ca(2+) channel. The association of endogenous sorcin and PS2 was demonstrated in cultured cells and human brain tissues. Membrane-associated sorcin and a subset of the functional PS2 complexes were co-localized to a novel subcellular fraction that is distinctively positive for calcineurin B. Sorcin was found to interact with PS2 endoproteolytic fragments but not full-length PS2, and the sorcin/PS2 interaction was greatly enhanced by treatment with the Ca(2+) ionophore A23187. Our findings reveal a molecular link between PS2 and intracellular Ca(2+) channels (i.e. ryanodine receptor) and substantiate normal and/or pathological roles of PS2 in intracellular Ca(2+) homeostasis.

Calcium-dependent binding of sorcin to the N-terminal domain of synexin (annexin VII).

The annexins are characterized by their ability to bind phospholipid membranes in a Ca2+-dependent manner. Sequence variability between the N-terminal domains of the family members may contribute to the specific cellular function of each annexin. To identify proteins that interact with the N-terminal domain of synexin (annexin VII), a fusion protein was constructed composed of glutathione S-transferase fused to amino acids 1-145 of human synexin. Affinity chromatography using this construct identified sorcin as a Ca2+-dependent synexin-binding protein. Overlay assays confirmed the interaction. The glutathione S-transferase construct associates with recombinant sorcin over the range of pCa2+ = 4.7-3.1 with no binding observed at pCa2+ = 5.4. Overlay assays using deletion constructs of the synexin N-terminal domain mapped the sorcin binding site to the N-terminal 31 amino acids of the synexin protein. Additionally, synexin forms a complex with sorcin and recruits this protein to chromaffin granule membranes in a Ca2+-dependent manner. Sorcin is able to inhibit synexin-mediated chromaffin granule aggregation in a manner saturable with increasing sorcin concentrations, but does not influence the Ca2+ sensitivity of synexin-mediated granule aggregation. Therefore, the interaction between sorcin and synexin may serve to regulate the functions of these proteins on membrane surfaces in a Ca2+-dependent manner.

Calcium-dependent binding of the plasma protein apolipoprotein A-I to two members of the annexin family.

Affinity chromatography with purified annexins coupled to CNBr-activated Sepharose 4B was used to determine the capacity of proteins found in cytosolic fractions of the bovine adrenal medulla to bind to an immobilized annexin in a Ca2+-dependent manner. Several proteins were eluted from a recombinant annexin I column in the presence of 2 mM EGTA, including protein kinase C (PKC), members of the annexin family, and a 26 kDa protein that appeared as the most prominent band on SDS-PAGE. The identities of PKC, annexin I, annexin IV, annexin VI, and annexin VII were confirmed by Western blotting. The 26 kDa protein was purified by anion exchange chromatography on a Poros Q column and determined to be apolipoprotein A-I (apoA-I) by peptide sequencing. Comigration of apoA-I and chromobindin 2 on two-dimensional gels identified apoA-I as chromobindin 2. Overlay assays were performed to verify the apoA-I-annexin I interaction using apoA-I immobilized on nitrocellulose and annexin I in solution with binding detected using anti-annexin I antiserum. Additionally, the ability of biotin-labeled apoA-I in solution to bind to several purified annexins immobilized on nitrocellulose was determined by detection with horseradish peroxidase-conjugated avidin. Using these methods, it was shown that both annexin I and annexin VII bind to bovine apoA-I in a Ca2+-dependent manner. Other annexins, such as annexin IV and annexin VI, do not exhibit this binding. The results suggest that certain annexins may function as extracellular binding sites for plasma proteins.