Dana Chuderland

Identification and Characterization of a General Nuclear Translocation Signal in Signaling Proteins

Upon stimulation, many proteins translocate into the nucleus in order to regulate a variety of cellular processes. The mechanism underlying the translocation is not clear since many of these proteins lack a canonical nuclear localization signal (NLS). We searched for an alternative mechanism in extracellular signal-regulated kinase (ERK)-2 and identified a 3 amino acid domain (SPS) that is phosphorylated upon stimulation to induce nuclear translocation of ERK2. A 19 amino acid stretch containing this phosphorylated domain inserts nondiffusible proteins to the nucleus autonomously. The phosphorylated SPS acts by binding to importin7 and the release from nuclear pore proteins. This allows its functioning both in passive and active ERK transports. A similar domain appears in many cytonuclear shuttling proteins, and we found that phosphorylation of similar sequences in SMAD3 or MEK1 also induces their nuclear accumulation. Therefore, our findings show that this phosphorylated domain acts as a general nuclear translocation signal (NTS).

Protein-protein interactions in the regulation of the extracellular signal-regulated kinase

The extracellular signal-regulated kinase (ERK) cascade is a central intracellular signaling pathway that is activated by a variety of extracellular stimuli, and thereby regulates cellular processes such as proliferation, differentiation, and oncogenic transformation. To execute these functions, the signals of those stimuli are transmitted to the cytosolic and nuclear targets in a rapid and specific manner. In the last few years it has become clear that the specificity and the rapid function of the ERK cascade is largely determined by protein-protein interactions with various signaling components and substrates. This review describes interactions of ERK with its immediate regulators, scaffold proteins, substrates, and localizing proteins, and shows their involvement in the functioning of the ERK cascade. Understanding the full scope of ERK-interactions is important for the development of new drugs for the treatment of cancer and other diseases.

Nuclear Extracellular Signal-Regulated Kinase 1 and 2 Translocation Is Mediated by Casein Kinase 2 and Accelerated by Autophosphorylation

ABSTRACT The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase [MAPK] family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. These results provide an important role of CK2 in regulating nuclear ERK activities.

Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration.

Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338-1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.

Calcium regulates ERK signaling by modulating its protein-protein interactions

Intracellular transmission of extracellular signals is mediated by a complex network of second messengers, interacting proteins and signaling cascades that cumulatively govern essentially all stimulated cellular processes. Two important signaling components in the network are the extracellular signal-regulated kinase (ERK) signaling cascade and elevation of calcium concentrations, which transmit signals of hormones, nerve factors and other ligands. Each of these components is known to independently regulate a large number of targets in various cellular organelles. The cooperation between them, however, seems to modulate the fate of their individual signals, and accordingly, their regulated processes. We have recently shown that calcium modulates the protein interaction properties of ERKs, which further affect the subcellular localization of the latter and as a consequence also the distribution of their targets. These effects of calcium are important in determining the specificity of the ERK cascade, and thereby, play important roles in the regulation of ERK-dependent cellular processes. Our findings, as well as their possible implications, are further discussed in this addendum. Addendum to: Chuderland D, Marmor G. Shainskaya A, Seger R. Calcium-mediated interactions regulate the subcellular localization of extracellular signal-regulated kinases. J Biol Chem 2008; 283:11176-88.

Calcium-mediated interactions regulate the subcellular localization of extracellular signal-regulated kinases.

The subcellular localization of ERKs in cells, which is important for proper signaling, may be regulated through protein-protein interactions. We found that inactive ERK2 interacts with a large number of proteins through its cytosolic retention sequence/common docking domain, whereas the phospho-ERK2 interacts with only few substrates. Varying calcium concentrations significantly modified the repertoire of ERK2-interacting proteins, of which many were identified. The effect of calcium on ERK interactions also influenced the localization of ERKs, as calcium chelators enhanced nuclear translocation, whereas elevated calcium levels prevented it. This effect of calcium was apparent upon lysophosphatidic acid stimulation, where ERKs translocation was delayed compared with that induced by EGF in a calcium-dependent manner. In vitro translocation assay revealed that high calcium concentrations affect ERK translocation by preventing the shuttling machinery through the nuclear envelope, probably due to higher binding to nuclear pore proteins. These results are consistent with a model in which ERKs in quiescent cells are bound to several cytoplasmic proteins. Upon stimulation, ERKs are phosphorylated and released from cytoplasmic anchors to allow shuttling toward the nucleus. This translocation is delayed when calcium levels are increased, and this modifies the localization of ERKs and, therefore, also their spatiotemporal regulation. Thus, calcium regulates ERK localization, which is important for the compartmentalization of ERKs with their proper substrates and thereby their signaling specificity.

Nuclear targeting sequences

Isolated peptides comprising nuclear targeting activity or being capable of preventing endogenous nuclear targeting activity are disclosed. Polynucleotides encoding same, pharmaceutical compositions comprising same, as well as uses thereof are also disclosed.