Emilio Itarte

Glycogen synthase: a new activity ratio assay expressing a high sensitivity to the phosphorylation state.

Since its introduction [I] the -glucose 6-phosphate/t glucose 6-phosphate (-G6P/tG6P) activity ratio assay for glycogen synthase has been a very useful tool in the study of the activation state of this enzyme. The idea of glycogen synthase in two forms (one active in the absence of added G6P (I form), the other requiring it for activity (D form)) evolved from the first experiments in which the -G6P/tG6P assay was employed [ 11. Shortly afterwards changes in the -G6P/tG6P activity ratio were correlated with phosphorylation and dephosphorylation of the enzyme [2]. At concentrations of metabolites close to their physiological levels it was shown [3] that the D form would be inactive whereas the I form would be active. Therefore conversion between D and I forms (i.e., changes in the -G6P/+G6P activity ratio) would correspond to changes in the ‘in vivo’ activity. Nevertheless, in physiological experiments rather small changes in the -G6P/tG6P activity ratio had been observed in response to the administration of hormones. These modest changes in the -G6P/tG6P activity ratio were rather hard to reconcile with an on-off control of glycogen synthase activity (reviewed [41). The enzyme has been shown to have a multiple phosphorylated subunit [5-l I] and that phosphorylation produces pronounced effects on the apparent affinity of the enzyme for its substrate UDP-glucose and the activator glucose 6-phosphate [7,11-131. In [ 131 we have demonstrated that when glycogen

Purification and characterization of two cyclic AMP-independent casein/glycogen synthase kinases from rat liver cytosol

Two cyclic AMP-independent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) (casein kinase 1 and 2) have been purified from rat liver cytosol by a method involving chromatography on phosphocellulose and casein-Sepharose 4B. Both kinases were essentially free of endogeneous protein substrates and capable of phosphorylating casein, phosvitin and I-form glycogen synthase, but were inactive on histone IIA, protamine and phosphorylase b. They were neither stimulated by cyclic AMP, Ca2+ and calmodulin, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. The casein and glycogen synthase kinase activities of each enzyme decreased at the same rate when incubated at 50 degrees C. Casein kinase 1 and casein kinase 2 showed differences in molecular weight, sensitivity to KCl, Km for casein and phosvitin and Ka for Mg2+, whereas their Km values for ATP and I-form glycogen synthase were similar. The phosphorylation of glycogen synthase by these kinases correlated with a decrease in the +/- glucose 6-phosphate activity ratio (independence ratio). However, casein kinase 1 catalyzed the incorporation of about 3.6 mol of 32P/85000 dalton subunit, decreasing the independence ratio from 83 to about 15, whereas the phosphorylation achieved by casein kinase 2 was only about 1.9 mol of 32P/850000 dalton subunit, decreasing the independence ratio to about 23. The independence ratio decrease was prevented by the presence of casein but was unaffected by phosphorylase b. These data indicate that casein/glycogen synthase kinases 1 and 2 are different from cyclic AMP-dependent protein kinase and phosphorylase kinase.

Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits

The acronym CK2 denotes a highly pleiotropic Ser/Thr protein kinase whose over‐expression correlates with neoplastic growth. A vexed question about the enigmatic regulation of CK2 concerns the actual existence in living cells of the catalytic (α and/or α′) and regulatory β‐subunits of CK2 not assembled into the regular heterotetrameric holoenzyme. Here we take advantage of novel reagents, namely a peptide substrate and an inhibitor which discriminate between the holoenzyme and the catalytic subunits, to show that CK2 activity in CHO cells is entirely accounted for by the holoenzyme. Transfection with individual subunits moreover does not give rise to holoenzyme formation unless the catalytic and regulatory subunits are co‐transfected together, arguing against the existence of free subunits in CHO cells.

PP1/PP2A phosphatases inhibitors okadaic acid and calyculin A block ERK5 activation by growth factors and oxidative stress

Okadaic acid is an inhibitor of the protein Ser/Thr phosphatases PP1 and PP2A, which blocks the activation of extracellular signal‐regulated protein kinase 5 (ERK5), a member of the MAP kinase family activated by growth factors and several types of stressors. The blocking of ERK5 activation by okadaic acid was observed in HeLa cells exposed to epidermal growth factor and H2O2 as well as in PC12 cells stimulated by nerve growth factor and H2O2. Calyculin A, another PP1 and PP2A inhibitor, behaved similarly although these compounds are not structurally related. This suggests that either PP1 or PP2A or both are necessary for ERK5 activation. Protein kinase C (PKC) acts as a negative regulator of the ERK5 activation pathway, however our data suggest that the effects of PKC and the phosphatase are unrelated.

Apigenin and LY294002 prolong EGF-stimulated ERK1/2 activation in PC12 cells but are unable to induce full differentiation.

In rat pheochromocytoma cell line (PC12) cells, initial epidermal growth factor (EGF)‐stimulated extracellular signal‐regulated protein kinases 1/2 (ERK1/2) phosphorylation was similar to that promoted by nerve growth factor (NGF), but declined rapidly. Pre‐treatment with apigenin or LY294002 sustained EGF‐stimulated ERK1/2 phosphorylation whereas wortmannin partially blocked initial ERK1/2 phosphorylation. Changes in ERK1/2 phosphorylation correlated with alterations in p90 ribosomal S6 kinase activity. Wortmannin, LY294002 and apigenin totally blocked growth factor‐induced protein kinase B phosphorylation. However, none of them potentiated Raf activation, which was in fact decreased by LY290042 and wortmannin. The sustained EGF‐induced ERK1/2 activation promoted by apigenin was not sufficient to commit PC12 cells to differentiate, which was achieved by stimulation with NGF, either alone or in the presence of apigenin.

The Regulatory β Subunit of Protein Kinase CK2 Contributes to the Recognition of the Substrate Consensus Sequence. A Study with an eIF2β-Derived Peptide

CK2 is a ubiquitous and pleiotropic Ser/Thr-specific protein kinase that phosphorylates more than 300 protein substrates at sites specified by an acidic consensus sequence in which positions n + 3 and n + 1 are particularly important. Recognition of substrates by CK2 is known to rely on basic residues located in the catalytic site of the alpha subunit which make electrostatic contacts with the negative charges in the substrate consensus sequence, thereby assuring optimal binding; the regulatory beta subunit is believed to play a protective and stabilizing role. We describe a biochemical and structural analysis of CK2-mediated phosphorylation of a 22-mer synthetic peptide corresponding to the N-terminal tail of the eukaryotic translation initiation factor eIF2beta. Results demonstrate that this peptide still displays phosphorylation features similar to full-length eIF2beta and the CK2 beta subunit also contributes to recognition of the protein substrate by establishing both polar and hydrophobic interactions with specificity determinants located downstream from the phosphoacceptor site. In particular, the N-terminal domain of the beta subunit appears to be of crucial importance for optimizing high-affinity phosphorylation of the eIF2beta peptide. This domain includes an acidic cluster whose electrostatic contacts with basic residues of the substrate attenuate intrasteric pseudosubstrate inhibition while strengthening substrate-kinase binding.

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.

Phosphorylation of hepatic insulin receptor by casein kinase 2.

Casein kinase 2 was able to phosphorylate the β‐subunit of hepatic insulin receptor in the presence of either ATP or GTP. Phosphorylation by casein kinase 2 was observed even in the absence of insulin, was inhibited by low heparin concentrations, and led to the incorporation of phosphate on serine and threonine residues. Casein kinase 2 phosphorylation of insulin receptor partially decreased its tyrosine kinase activity.

Casein kinase 2 activity increases in the prereplicative phase of liver regeneration

Cytosolic casein kinase activity increased up to 2‐fold in the first 6 h after partial hepatectomy and then decreased to control values. This increase was due mainly to casein kinase 2, which reached maximal values at 6–8 h of liver regeneration. In contrast, casein kinase 1 showed a smaller increase at 4 h and then started to decrease reaching values of about 70% of control at 16 h. The increase in total casein kinase 2 was accompanied with an activation of the enzyme, as determined by the low/high β‐casein activity ratio assay. Administration of an acute dose of glucagon to control rats also increased the activity ratio but failed to cause any rise in total casein kinase 2 activity.

Proteomic analysis of SET-binding proteins.

The protein SET is involved in essential cell processes such as chromatin remodeling, apoptosis and cell cycle progression. It also plays a critical role in cell transformation and tumorogenesis. With the aim to study new SET functions we have developed a system to identify SET‐binding proteins by combining affinity chromatography, MS, and functional studies. We prepared SET affinity chromatography columns by coupling the protein to activated Sepharose 4B. The proteins from mouse liver lysates that bind to the SET affinity columns were resolved with 2‐DE and identified by MS using a MALDI‐TOF. This experimental approach allowed the recognition of a number of SET‐binding proteins which have been classified in functional clusters. The identification of four of these proteins (CK2, eIF2α, glycogen phosphorylase (GP), and TCP1‐β) was confirmed by Western blotting and their in vivo interactions with SET were demonstrated by immunoprecipitation. Functional experiments revealed that SET is a substrate of CK2 in vitro and that SET interacts with the active form of GP but not with its inactive form. These data confirm this proteomic approach as a useful tool for identifying new protein–protein interactions.