Francesc Miró

Osmotic stress regulates the stability of cyclin D1 in a p38SAPK2-dependent manner.

We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38SAPK2-specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38SAPK2activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38SAPK2-dependent pathway. The effect of p38SAPK2 on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38SAPK2 phosphorylates cyclin D1 in vitroat Thr286 and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.

Eukaryotic translation-initiation factor eIF2beta binds to protein kinase CK2: effects on CK2alpha activity.

eIF2 (eukaryotic translation-initiation factor 2) is a substrate and an interacting partner for CK2 (protein kinase CK2). Co-immuno-precipitation of CK2 with eIF2beta has now been observed in HeLa cells, overexpressing haemagglutinin-tagged human recombinant eIF2beta. A direct association between His6-tagged human recombinant forms of eIF2beta subunit and both the catalytic (CK2alpha) and the regulatory (CK2beta) subunits of CK2 has also been shown by using different techniques. Surface plasmon resonance analysis indicated a high affinity in the interaction between eIF2beta and CK2alpha, whereas the affinity for the association with CK2beta is much lower. Free CK2alpha is unable to phosphorylate eIF2beta, whereas up to 1.2 mol of phosphate/mol of eIF2beta was incorporated by the reconstituted CK2 holoenzyme. The N-terminal third part of eIF2beta is dispensable for binding to either CK2alpha or CK2beta, although it contains the phosphorylation sites for CK2. The remaining central/C-terminal part of eIF2beta is not phosphorylated by CK2, but is sufficient for binding to both CK2 subunits. The presence of eIF2beta inhibited CK2alpha activity on calmodulin and beta-casein, but it had a minor effect on that of the reconstituted CK2 holoenzyme. The truncated forms corresponding to the N-terminal or central/C-terminal regions of eIF2beta were much less inhibitory than the intact subunit. The results demonstrate that the ability to associate with CK2 subunits and to serve as a CK2 substrate are confined to different regions in eIF2beta and that it may act as an inhibitor on CK2alpha.

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2α/α′ antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2.

Cloning and Characterization of CD300d, a Novel Member of the Human CD300 Family of Immune Receptors

Background: Leukocyte function is regulated by the balance between activating and inhibitory signals deliver by immune receptors. Results: We present the cloning and molecular characterization of a novel CD300 member, CD300d. Conclusion: The function of CD300d is related to the regulation of the expression and function of other CD300 receptors. Significance: A new mechanism of regulation of myeloid cell activation is described. Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with the CD300 family members, with the exception of CD300c. Contrary to other activating members of the CD300 family of receptors, surface expression of CD300d in COS-7-transfected cells required the presence of an immunoreceptor tyrosine-based activating motif-bearing adaptor (FcϵRγ). Accordingly, we found that CD300d was able to recruit FcϵRγ. Unexpectedly, we could not detect CD300d on the surface of cells expressing FcϵRγ, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules also did not modify the intracellular expression of CD300d. In fact, the presence of CD300d decreased the levels of surface expression of CD300f but not CD300c. Our data suggest that the function of CD300d would be related to the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.

The carboxy‐terminal domain of Grp94 binds to protein kinase CK2α but not to CK2 holoenzyme

Surface plasmon resonance analysis shows that the carboxy‐terminal domain of Grp94 (Grp94‐CT, residues 518–803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2α) under non‐stressed conditions. A K D of 4×10−7 was determined for this binding. Heparin competed with Grp94‐CT for binding to CK2α. CK2β also inhibited the binding of Grp94‐CT to CK2α, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94‐CT. The use of CK2α mutants made it possible to map the Grp94‐CT binding site to the four lysine stretch (residues 74–77) present in helix C of CK2α. Grp94‐CT stimulated the activity of CK2α wild‐type but was ineffective on the CK2α K74–77A mutant.

Persistent nuclear accumulation of protein kinase CK2 during the G1-phase of the cell cycle does not depend on the ERK1/2 pathway but requires active protein synthesis.

Protein kinase CK2 and phosphorylated ERK1/2 accumulated in nucleus after serum stimulation of quiescent HepG2 cells. Nonetheless, phospho-ERK1/2 accumulated mainly in the nuclease-extracted fraction (NE) whereas the increases in nuclear CK2 (either CK2alpha or CK2beta) occurred initially in the nuclease-resistant fraction (NR). Transient decreases in CK2 were observed in cytoplasm and NE in the first 3h but thereafter they either reverted (cytoplasm) or increased above the control (NE). CK2 levels in both NE and NR were high in cells arrested at G1/S. Maximal nuclear accumulation of CK2 was blocked by cycloheximide but little affected by PD98059, SB203580 or apigenin, all of which affected nuclear phopho-ERK1/2. Thus, nuclear accumulation of CK2 during G1 phase is independent of ERK1/2 pathway. Although this process may initially relay on intracellular redistribution of the preexisting enzyme, active protein synthesis is required to attain maximal nuclear CK2 levels.

Tumour suppressor protein p53 released by nuclease digestion increases at the onset of rat liver regeneration

BACKGROUND/AIMS Protein kinase CK2 (CK2) increases when cells are committed to proliferate, as in liver regeneration. This enzyme phosphorylates the tumour suppressor protein p53, whose expression controls the levels of many other cell cycle proteins. The aim of this study was to determine if CK2 was affected by p53. METHODS Male Sprague-Dawley rats (200-250 g) were subjected to either partial hepatectomy or laparotomy and the levels and subcellular distribution of p53 were studied, following the approach used earlier for CK2. The levels of both proteins were also studied in the human cell lines HL-60 (devoid of p53) and HepG2 (with normal p53 levels) and in fibroblasts from transgenic p53-deficient mice (p53-/-) or homozygous for wild-type p53 (p53+/+). Computer-assisted search was used to detect p53 consensus sequences in genes for CK2 subunits Binding of p53 protein to some of these sequences was assayed by electrophoretic mobility shift assay. RESULTS Rat liver p53 protein was present mainly in the fraction extracted from intact nuclei by nucleases (S1) and showed a transient increase at 6 h post partial hepatectomy, as observed previously with nuclear CK2. The human CK2a gene presents the consensus sequence for trans-activation by p53 and specific binding of p53 protein to some of these sequences was detected in vitro. Total CK2a was higher in HepG2 than in HL-60 cells but total CK2 and its cytosolic/ nuclear distribution was similar in mice (p53+/+) fibroblasts and (p53-/-) fibroblasts. CONCLUSIONS p53 is present in the nuclease-extracted S1 fraction from liver cells, as described for CK2, and undergoes similar changes at the beginning of rat liver regeneration. However, the data on cultured cells suggest that the expression of CK2 and its subcellular localization are p53-independent events.

Protein kinase CK2 is altered in insulin-resistant genetically obese (fa/fa) rats

Hepatic insulin receptor levels in 6‐week‐old obese (fa/fa) rats were about 2‐fold lower than those from lean (Fa/−) rats, which agrees with their insulin‐resistant state. Nuclear protein kinase CK2 activity and protein content in livers from obese (fa/fa) rats were similar to those of lean (Fa/−) animals but the cytosolic levels were reduced to half, due to a decrease in the 39‐kDa catalytic subunit. Marked increases in activity, due to rises in the 44‐kDa and 39‐kDa catalytic subunits, were seen in the 16 000×g sediments (M1) from insulin‐resistant rats, with moderate changes in the 100 000×g sediments (M2). The increase in CK2 binding to M1 did not require increases in the molecular chaperone grp94, which was unaltered in insulin‐resistant rats.