Germaine Jacob

Stereospecificity of isopentenylpyrophosphate isomerase and prenyl transferase from Pinus and Citrus

Abstract Isopentenylpyrophosphate isomerases in cell-free extracts from Pinus radiata and from Citrus sinensis eliminate the C-4 pro-S proton from mevalonic acid. Prenyl transferase from Pinus forms geranyl pyrophosphate, neryl pyrophosphate, 2,6 trans, trans farnesyl pyrophosphate and 2 cis, 6 trans farnesyl pyrophosphate from mevalonic acid. The 4-pro-S proton of mevalonic acid is eliminated in the formation of both trans and of cis double-bonds. The pattern is similar for the Citrus enzyme. Isopentenyl pyrophosphate isomerase from Pinus is completely inhibited by 15 m m iodoacetamide, which does not affect prenyl transferase. The pattern is the same for the Citrus isomerase, but less clear for the transferase. Previous work has ruled out direct trans-cis isomerization of C10 or C15 prenylpyrophosphates. Alternative mechanisms are proposed for the stereospecific elimination of the 4-pro-S proton of mevalonic acid in the biosynthesis of both cis and trans prenyl pyrophosphates.

Autophosphorylation of carboxy‐terminal residues inhibits the activity of protein kinase CK1α

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1δ and CK1ε were previously shown to autophosphorylate carboxy‐terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1α and splice variant CK1αL has identified the autophosphorylation of the last four carboxyl‐end serines and threonines and also for CK1αS, the same four residues plus threonine‐327 and serine‐332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy‐terminal phosphorylation sites of CK1α to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1α and CK1αS with λ phosphatase causes an activation of 80–100% and 300%, respectively. Similar treatment fails to stimulate the CK1α mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1α and CK1αS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1α and CK1αS was also observed using the known CK1 protein substrates DARPP‐32, β‐catenin, and CK2β, which have different CK1 recognition sequences. Autophosphorylation effects on CK1α activity are not due to changes in Kmapp for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1α and its splice variants can be regulated by their autophosphorylation status. J. Cell. Biochem. 106: 399–408, 2009.

Synthesis of isomeric farnesols by soluble enzymes from Pinus radiata seedlings

Abstract Water soluble enzymes obtained from Pinus radiata seedlings form two sesquiterpene alcohols from 2- 14 C mevalonic acid. They have been identified as 2,6- trans,trans -farnesol and 2- cis ,6- trans -farnesol. The pyrophosphate of the former prenol could be isolated, but there was no evidence of the presence of phosphorylated derivatives of the cis isomer. The same pair of sesquiterpene alcohols and trans -farnesyl pyrophosphate are formed from isopentenyl pyrophosphate plus geranyl pyrophosphate (2- trans ). Neryl pyrophosphate (2- cis ) is completely inactive as a precursor of farnesols. Isomerization of trans - to cis -farnesyl pyrophosphate did not occur in this system.

Role of the carboxyl terminus on the catalytic activity of protein kinase CK2α subunit

Protein kinase CK2 (also known as casein kinase 2) has catalytic (α, α′) and regulatory (β) subunits. The role of carboxyl amino acids in positions from 324 to 328 was studied for Xenopus laevis CK2α. Deletions and mutations of these residues were produced in recombinant CK2α, which was assayed for kinase activity. Activity dropped 7000‐fold upon deletion of amino acids 324–328. The key residues are isoleucine 327 and phenylalanine 324. A three dimensional model of CK2α indicates that these hydrophobic residues of helix αN may interact with hydrophobic residues in helix αE which is linked to the catalytic center.

The activity of CK2 in the extracts of COS-7 cells transfected with wild type and mutant subunits of protein kinase CK2.

Protein kinase CK2 is ubiquitous in eukaryotes and is known to phosphorylate many protein substrates. The enzyme is normally a heterotetramer composed of catalytic (α and α′) and regulatory (β) subunits. The physiological regulation of the enzyme is still unknown but one of the factors that may play an important role in this regulation is the ratio of the catalytic and regulatory subunits present in cells. The possible existence of ‘free’ CK2 subunits, not forming part of the holoenzyme, may be relevant to the physiological function of the enzyme in substrate selection or in the interaction of the subunits with other partners. The objective of this work was to study in COS-7 cells the effects of transient expression of CK2 subunits and mutants of the catalytic subunit on the CK2 phosphorylating activity of the extracts of these cells. Using pCEFL vectors that introduce hemaggutinin (HA) or a heptapeptide (AU5) tags in the expressed proteins, COS-7 cells were transfected with α and β subunits of Xenopus CK2, with the α′ subunit of D. rerio, and with Xl CK2αA156, which although inactive can bind tightly to CK2β, and with Xl CK2αE75E76, which is resistant to heparin and polyanion inhibition. The efficiency of transient transfection was of 10–20% of treated cells.Expression of CK2α or CK2αE75E76 in COS-7 cells caused an increase of 5–7-fold of the CK2 activity in the soluble cell extracts. If these catalytic subunits were cotransfected with CK2β, the activity increased further to 15–20-fold of the controls. Transfection of CK2β alone also increase the activity of the extracts about 2-fold. Transfection with the inactive CK2αA156 yielded extracts with CK2 activities not significantly different from those transfected with the empty vectors. However, cotransfection of CK2α or CK2αE75E76 with CK2αA156 caused a 60–70% decrease in the CK2 activity as compared to those of cells transfected with only the active CK2α subunits. These results can be interpreted as meaning that CK2αA156 is a dominant negative mutant that can compete with the other catalytic subunits for the CK2β subunit. Addition of recombinant CK2β to the assay system of extracts of cells transfected with catalytic subunits causes a very significant increase in their CK2 activity, demonstrating that CK2β subunit is limiting in the extracts and that an excess of free CK2α has been produced in the transfected cells. Transfection of cells with CK2αE75E76 results in a CK2 activity of extracts that is 90% resistant to heparin demonstrating that a very large proportion of the CK2 activity is derived from the expression of the exogenous mutant. In both the in vivo and in vitro systems, the sensitivity of CK2αE75E76 to heparin increases considerably when it forms part of the holoenzyme CK2α2β2.

Dual effect of lysine-rich polypeptides on the activity of protein kinase CK2.

Protein kinase CK2 (casein kinase II) is normally a heterotetramer composed of catalytic (α, α′) and regulatory subunits (β). CK2 is able to phosphorylate a large number of protein substrates but the physiological mechanisms of its regulation are still unresolved. Lysine‐rich peptides such as polylysine and histone H1 are known to stimulate the catalytic activity of the holoenzyme. This activation is mediated through the CK2β regulatory subunit. In this communication, we report that the same concentrations of lysine‐rich peptides or proteins that activate the holoenzyme cause strong inhibition of the phosphorylation of proteins catalyzed by the free catalytic CK2α subunit. The inhibitory effect of polylysine and histone H1 is observed with several protein substrates of CK2α (casein, adeno E1A, transcription factor II A, and CK2β itself). With calmodulin, however, the inhibition of CK2α phosphorylation caused by polylysine is much lower while with a model peptide substrate of CK2 the inhibition caused by this polycation is negligible. The inhibition of CK2α by polylysine is observed only at limiting concentrations of the target substrate proteins. The dual effect of polylysine and of histone H1, which results in the inhibition of CK2α and stimulation of the CK2 α2β2 tetrameric holoenzyme, has the consequence that the addition of the CK2β, in the presence of polylysine and low concentrations of substrate protein, can cause a 242‐fold stimulation of the activity of CK2α. Other polycationic compounds such as polyarginine and spermine do not inhibit the phosphorylation of casein by CK2α, indicating that the effect is specific for lysine‐rich peptides. Since there is evidence that there may be free CK2α subunits in the nuclei of cells, where there is abundant histone H1, the inhibition of CK2α by this lysine‐rich protein may have physiological relevance. J. Cell. Biochem. 89: 348–355, 2003.

Characteristics of phospholipase C present in membranes of Xenopus laevis oocytes. Stimulation by phosphatidic acid

1. Phospholipase C activity present in the membranes of Xenopus laevis oocytes has been studied. 2. These membranes contain an activity capable of hydrolyzing phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. 3. Hydrolysis of PtdIns 4,5-P2 is absolutely dependent on the presence of Ca2+, however, the hydrolysis of PtdIns occurs in the absence of Ca2+ but addition of the cation stimulates the reaction. 4. Spermine, spermidine and polylysine cause significant stimulation of the phospholipase C activity. 5. Phosphatidic acid causes approximately a 2-fold stimulation of the hydrolysis of both PtdIns and PtdIns 4,5-P2. With PtdIns as substrate, this stimulatory effect of phosphatidic acid is specific and reaches a maximum at a 400 microM concentration.

Involvement of asparagine 118 in the nucleotide specificity of the catalytic subunit of protein kinase CK2

Protein kinase CK2 is a heteromeric enzyme with catalytic (α) and regulatory (β) subunits which form an α2β2 holoenzyme and utilizes both ATP and GTP as nucleotide substrate. Site‐directed mutagenesis of CK2α subunit was used to study this capacity to use GTP. Deletion of asparagine 118 (αΔN118) or the mutant αN118E gives a 5–6‐fold increase in apparent K m for GTP with little effect on the affinity for ATP. Mutants αN118A and αD120N did not alter significantly the K m for either nucleotide. CK2αΔN118 has an apparent K i for inosine 5′ triphosphate 5‐fold higher than wild‐type and is very heat labile. These studies complement recent crystallographic data indicating a role for CK2α asparagine 118 in binding the guanine base.

The hydrolysis of phosphatidylinositol 4-phosphate in membranes of Xenopus laevis oocytes: Characteristics of a phosphomonoesterase

1. Phosphatidylinositol 4-phosphate (PtdIns4P) is degraded by isolated membranes from Xenopus laevis oocytes. 2. Incubation of [4-32P]PtdIns4P with membranes yields only radioactive inorganic phosphate, indicating the presence of a phosphomonoesterase. 3. Membranes hydrolyze Ptd[2-3H]Ins4P to produce mainly Ptd[2-3H]Ins in the lipid phase. In this incubation [3H]inositol and inositol monophosphate appear in the water phase. 4. Membrane incubations of Ptd[2-3H]Ins4P carried out in the presence of excess non-radioactive Ins(1,4)P2 allows the trapping of small amounts of [3H]Ins(1,4)P2. These results demonstrate the presence of a phospholipase C. 5. Testing several phosphorylated analogs, it is determined that fructose 1,6-bisphosphate and alpha-glycerophosphate are potent inhibitors of the oocyte PtdIns4P phosphomonoesterase.