Namely, in the first case, AnxA2 was immunoprecipitated from purified mRNP complexes formed by its cognate mRNA, while in the latter case, AnxA2 was immunoprecipitated from a subcellular fraction containing not only proteins present in mRNP complexes but also residing in additional cellular structures. related to the mechanisms that regulate the spatio\temporal dynamics of the maturing mRNP complexes 37. Here we present fresh evidence showing that Ser25\phosphorylated high\molecular\mass forms of AnxA2 C which are also ubiquitinated and/or sumoylated C associate with nonpolysomal mRNP complexes that look like enriched in the perinuclear region of Personal computer12 cells. Furthermore, inhibition of PKC inhibits the Ser25 phosphorylation of AnxA2 and prevents its localisation to the perinuclear region and results in the enrichment of AnxA2 in the inner cortical region of the plasma membrane. Results and conversation Subcellular localisation of pSer25AnxA2 We previously showed the phospho\mimicking AnxA2\Ser25Glu mutant and AnxA2 Ser25 phosphorylated by PKC are not targeted to the nucleus. Furthermore, the phospho\mimicking mutant displayed an eIF4A3-IN-1 increased affinity for mRNA studies have shown that phosphorylation of Ser25 increases the direct association of AnxA2 with mRNA 16, but the relevance of this getting has not been previously tackled. Consequently, the nuclear portion (including NE) enriched in pSer25AnxA2 (Fig. ?(Fig.1,1, lane 7) was further subfractionated into the corresponding polysomal and nonpolysomal populations containing translationally active and inactive mRNAs respectively (Fig. ?(Fig.4A,B).4A,B). The nuclear polysomal portion consists of no AnxA2 and only negligible amounts of the high\molecular\mass bands of pSer25AnxA2 (Fig. ?(Fig.4B,4B, lane 5) associated with poly(A)\containing mRNAs (Fig. ?(Fig.4B,4B, lane 6), in accordance with previous results showing the 39 kDa form of AnxA2 associates with cytoskeleton\bound polysomes 28. This indicates that pSer25AnxA2 is not involved in active mRNA translation in the cytoplasmic (results not demonstrated) or NE\connected polysomes. By contrast, pSer25AnxA2 and S6 kinase are enriched in the oligo(dT)\isolated nonpolysomal mRNP complexes, as compared to the starting portion (Fig. ?(Fig.4B,4B, compare lanes 2 and 4), while S6, a marker of small ribosomal subunits, is enriched in the nuclear polysomal pellet (Fig. ?(Fig.4B,4B, lane 5). This indicates the specificity of the connection of pSer25AnxA2 with nonpolysomal, NE\connected mRNP complexes present in the nuclear portion (Fig. ?(Fig.2).2). Therefore, pSer25AnxA2 could be involved in mRNA transport and/or sequestering of inactive mRNAs in mRNP complexes, most likely in P\body and/or stress granules 46, 47. The finding that AnxA2 binds to the localisation signal in the 3UTR of c\mRNA 30, which focuses on the mRNA to the perinuclear eIF4A3-IN-1 region for subsequent translation 36, is definitely consistent with this idea. Open in a separate window Number 4 pSer25AnxA2 and ubiquitinated high\molecular\mass forms of AnxA2 associate with translationally inactive mRNP complexes. (A, B) Large\molecular\mass forms of pSer25AnxA2 is present in oligo(dT)\purified nonpolysomal mRNP complexes in Personal computer12 cells. (A) Schematic representation of the method used in (B) with reference to the individual lanes in (B). (B) Samples (100 g of protein) were prepared from the following fractions: nucleus (lane 1), supernatant (lane 2) and polysome\comprising pellet (lane 5; derived from the nuclear portion after centrifugation for 2 h 100 000 through a 1 m sucrose cushioning), non\oligo(dT)\bound supernatant (lane 3), oligo(dT)\bound supernatant (lane 4), and oligo(dT)\bound Rabbit Polyclonal to EPHA3/4/5 (phospho-Tyr779/833) pellet (lane 6), as indicated above the western blot. The proteins were separated by 10% SDS/PAGE and subjected to western blot analysis. The blots were probed with antibodies against pSer25AnxA2, total AnxA2, tubulin, SPC, S6 kinase and the ribosomal protein S6, as indicated. Detection of the producing protein bands was performed from the ChemiDoc? XRS+ molecular imager eIF4A3-IN-1 after incubation with HRP\conjugated secondary antibodies and ECL reagent. The arrowheads to the left indicate the protein molecular mass requirements. (C, D) Large\molecular\mass forms of AnxA2 in mRNP complexes affinity\purified via binding to transcribed and polyadenylated full\length experiments failed to resolve this query 25. The protein has been reported to have a relatively long half\existence (~ 15 h) and to become degraded by chaperone\mediated autophagy 49, arguing against this possibility. To gain further insight into the practical role of the pSer25AnxA2\comprising nonpolysomal mRNP complexes, double\localisation studies with markers of P\body (GW182), stress granules (TIA\1) and neuronal granules (HuD), which all eIF4A3-IN-1 consist of translationally inactive mRNAs, were performed. Of the three marker proteins, pSer25AnxA2 only showed partial colocalisation with the P\body marker GW182 (Fig. ?(Fig.5,5, arrows; observe also intensity profiles). Arsenite treatment did not increase its colocalisation with any of the markers (data not shown). These studies further suggest that pSer25AnxA2 associates primarily with actively transferred mRNP complexes, rather than contributing to the sequestration of the connected mRNAs in or next to P\body, either for transient storage or degradation. Previous results showing that markers of P\body and transferred RNPs do not colocalise in the dendrites of adult hippocampal neurons lead to the proposal that dendritic mRNAs could.