Phosphorylation of Rat Liver Heterogeneous Nuclear Ribonucleoproteins A2 And C Can be Modulated by Calmodulin

Abstract

). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36-and 40-to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca 2؉ -dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., ␤-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca 2؉ -dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei. Calmodulin (CaM) is a ubiquitous Ca 2ϩ-binding protein which has been highly conserved during evolution. The binding of Ca 2ϩ to CaM induces a conformational change which permits the interaction of CaM with specific target proteins, i.e., CaM-binding proteins. During the last few years, compelling evidence indicating that CaM is present in the cell nucleus (4) and that it has a role in several nuclear functions, such as gene transcription (21), DNA replication, DNA repair, and mitosis (see references 3, 4, and 36 for reviews), has accumulated.One of the ways to understand the molecular mechanisms by which CaM regulates these nuclear functions has been the identification of its nuclear protein targets. At least three groups of CaM-binding proteins have been found in the nuclei of different cell types: (i) proteins related to the actin motility systems, e.g., ␣-spectrin, myosin light-chain kinase, and caldesmon (6, 51, 57); (ii) transcription factors belonging to the basic helix-loop-helix structural group, e.g., human SEF-1, mouse E2A, and Max (21); and (iii) proteins involved in the phosphorylation or dephosphorylation of proteins, e.g., CaM-dependent protein kinase II and the CaM-dependent protein phosphatase calcineurin (11,51,54). The role of the specific complexes of CaM with these CaM-binding proteins in the nuclear functions still remains obscure, although some possible pathways have recently emerged. It has recently been reported that the addition of antiactin antibodies blocks the transport of macromolecules into purified nuclei in in vitro experiments, suggesting that the nuclear actin-myosin contractile system can play a role in nucleocytoplasmic transport (55). The transcription factors which are able to bind CaM and belong to the basic helix-loop-helix grou...

Control of alternative pre-mRNA splicing by Ca++ signals

“…In an experiment using calmodulin-affinity chromatography, several RNA binding proteins including hnRNP A2 and C were identified [190]. The binding of CaM to these splicing factors inhibits their phosphorylation by Casein kinase II [190].…”

“…In an experiment using calmodulin-affinity chromatography, several RNA binding proteins including hnRNP A2 and C were identified [190]. The binding of CaM to these splicing factors inhibits their phosphorylation by Casein kinase II [190]. SAP145 [191], a member of the spliceosome-associated proteins and a subunit of SF3 (b) [49], was also identified as binding to a CaM affinity column.…”

Heterogeneous nuclear ribonucleoprotein A2 is a SET-binding protein and a PP2A inhibitor

“…This column was prepared by coupling purified recombinant human SET protein to Sepharose 4B beads. HnRNPA2 purified from mouse liver nuclei (Bosser et al, 1995) was loaded onto a SETSepharose 4B column or onto a Sepharose 4B column that was used as a control. Then, the binding of hnRNPA2 to the columns was analysed by Western blotting using anti-hnRNPA2 antibodies.…”

Isolation of Protein Subpopulations Undergoing Protein-Protein Interactions

“…4). Most of the remaining identifiable spots, including ATP synthase, mitochondrial ATPase inhibitor, and heterogeneous nuclear ribonucleoprotein A2, are also either known calcium-binding proteins or proteins that interact directly with calcium-binding proteins (22)(23)(24)(25)(26). Peptides from S100, another calcium-binding protein that undergoes numerous calcium-dependent protein interactions (27), were also detected.…”