Shin-ichi Imai

CLONING AND CHARACTERIZATION OF TWO HUMAN ISOZYMES OF MG2+-INDEPENDENT PHOSPHATIDIC ACID PHOSPHATASE

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (M r = 32,158) and -2b (M r = 35, 119), both of which contained six putative transmembrane domains. Both enzymes were glycosylated and cleaved by N-glycanase and endo-β-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b shared 47% identical sequence and were judged to be the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34–39% identical to that of DrosophilaWunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyzed lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation.

Identification and cDNA Cloning of 35-kDa Phosphatidic Acid Phosphatase (Type 2) Bound to Plasma Membranes POLYMERASE CHAIN REACTION AMPLIFICATION OF MOUSE H2O2-INDUCIBLE hic53 CLONE YIELDED THE cDNA ENCODING PHOSPHATIDIC ACID PHOSPHATASE

We previously described the purification of an 83-kDa phosphatidic acid phosphatase (PAP) from the porcine thymus membranes (Kanoh, H., Imai, S.-i., Yamada, K. and Sakane, F. (1992) J. Biol. Chem. 267, 25309-25314). However, we found that a minor 35-kDa protein could account for the PAP activity when the purified enzyme preparation was further analyzed. We thus determined the N-terminal sequence of the 35-kDa candidate protein and prepared antipeptide antibody against the determined sequence, MFDKTRLPYVALDVL. The antibody almost completely precipitated the purified enzyme activity. Furthermore, the antibody precipitated from the radioiodinated enzyme preparation a single 35-kDa protein, which was converted to a 29-kDa form when treated with N-glycanase. We also found that the immunoprecipitable PAP activity was exclusively associated with the plasma membranes of porcine thymocytes. These results indicated that the 35-kDa glycosylated protein represents the plasma membrane-bound (type 2) PAP. We surprisingly noted that the N-terminal sequence of the porcine PAP was almost completely conserved in the internal sequence encoded by a mouse partial cDNA clone, hic53, reported as a H2O2-inducible gene (Egawa, K., Yoshiwara, M., Shibanuma, M., and Nose, K. (1995) FEBS Lett. 372, 74-77). We thus amplified from the mouse kidney RNA the hic53 clone by polymerase chain reaction, and obtained a cDNA encoding a novel protein of 283 amino acid residues with a calculated Mr of 31,894. Methionine reported as an internal residue was found to serve as an initiator, and the C-terminal 64 residues were lacking in hic53. The protein contains several putative membrane-spanning domains and two N-glycosylation sites. When transfected into 293 cells, the cDNA gave more than 10-fold increase of the membrane-bound PAP activity, which could be precipitated by the antipeptide antibody. In [35S]methionine-labeled cells, the translational product was confirmed to be a 35-kDa protein, which became 30 kDa in cells treated with tunicamycin, an inhibitor of N-glycosylation. We thus succeeded first in identifying the porcine type 2 PAP and subsequently in determining the primary structure of a mouse homolog of the PAP.

Identification and Characterization of a Novel Human Type II Diacylglycerol Kinase, DGKκ

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Here we identified a tenth member of the DGK family designated DGKκ. The κ-isozyme (1271 amino acids, calculated molecular mass, 142 kDa) contains a pleckstrin homology domain, two cysteine-rich zinc finger-like structures, and a separated catalytic region as have been found commonly for the type II isozymes previously cloned (DGKδ and DGKη). The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N terminus. Reverse transcriptase-PCR showed that the DGKκ mRNA is most abundant in the testis, and to a lesser extent in the placenta. DGKκ, when expressed in HEK293 cells, was persistently localized at the plasma membrane even in the absence of cell stimuli. Deletion analysis revealed that the short C-terminal sequence (amino acid residues 1199–1268) is necessary and sufficient for the plasma membrane localization. Interestingly, DGKκ, but not other type II DGKs, was specifically tyrosine-phosphorylated at Tyr78 through the Src family kinase pathway in H2O2-treated cells. Moreover, H2O2 selectively inhibited DGKκ activity in a Src family kinase-independent manner, suggesting that the isozyme changes the balance of signaling lipids in the plasma membrane in response to oxidative stress. The expression patterns, subcellular distribution, and regulatory mechanisms of DGKκ are distinct from those of DGKδ and DGKη despite high structural similarity, suggesting unique functions of the individual type II isozymes.

Promotion of transferrin folding by cyclic interactions with calnexin and calreticulin

Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum. Because they have an affinity for monoglucosylated N‐linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding. To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells. Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution. In this system, addition of a glucose donor, UDP‐glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP‐glucose enhanced the amount of folded transferrin and reduced the disulfide‐linked aggregates. Analysis of transferrin folding in briefly heat‐treated microsomes revealed that UDP‐glucose was also effective in elimination of heat‐induced misfolding. Incubation of the microsomes with an α–glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex. Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.

Identification and Characterization of Two Splice Variants of Human Diacylglycerol Kinase η

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKη (η1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGKη isoform (η2, 135 kDa) that shared the same sequence with DGKη1 except for a sterile α motif (SAM) domain added at the carboxyl terminus. The DGKη1 mRNA was ubiquitously distributed in various tissues, whereas the DGKη2 mRNA was detected only in testis, kidney, and colon. The expression of DGKη2 was suppressed by glucocorticoid in contrast to the marked induction of DGKη1. DGKη2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (δ1 and δ2). Interestingly, DGKη1 and DGKη2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGKη1 was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGKη2 exhibited sustained endosomal association. The experiments using DGKη mutants suggested that the oligomerization of DGKη2 mediated by its SAM domain was largely responsible for its sustained endosomal localization. Similarly, the oligomerization of DGKη2 was suggested to result in negative regulation of its catalytic activity. Taken together, alternative splicing of the human DGKη gene generates at least two isoforms with distinct biochemical and cell biological properties responding to different cellular metabolic requirements.

Regulation of Enzyme Localization by Polymerization: Polymer Formation by the SAM Domain of Diacylglycerol Kinase δ1

The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.

Phorbol Ester-regulated Oligomerization of Diacylglycerol Kinase δ Linked to Its Phosphorylation and Translocation

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKδ containing a sterile α-motif (SAM) domain. We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures. Moreover, gel filtration and co-immunoprecipitation analyses demonstrated that DGKδ formed homo-oligomeric structures in intact cells and that the SAM domain was critically involved in the oligomerization. Interestingly, phorbol ester stimulation induced dissociation of the oligomeric structures with concomitant phosphorylation of DGKδ. Furthermore, we found that DGKδ was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation. In this case, DGKδ mutants lacking the ability of self-association were localized at the plasma membranes even in the absence of phorbol ester. A protein kinase C inhibitor, staurosporine, blocked all of the effects of phorbol ester,i.e. oligomer dissociation, phosphorylation, and translocation. We confirmed that tumor-promoting phorbol esters did not directly bind to DGKδ. The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKδ and that DGKδ is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.

The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the pleckstrin homology domain

DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.

Diacylglycerol Kinase η Augments C-Raf Activity and B-Raf/C-Raf Heterodimerization

The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase η (DGKη) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKη1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKη serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKη-silenced HeLa cells, this study revealed that DGKη activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKη enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKη physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKη-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKη acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.

Diacylglycerol Kinases as Emerging Potential Drug Targets for a Variety of Diseases

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating (consuming) DAG to yield PA. Ten mammalian DGK isozymes have been identified to date. In addition to two or three cysteine-rich C1 domains (protein kinase C-like zinc finger structures) commonly conserved in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain, a MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation site domain and ankyrin repeats. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of mammalian signal transduction pathways conducting growth factor/cytokine-dependent cell proliferation and motility, seizure activity, immune responses, cardiovascular responses and insulin receptor-mediated glucose metabolism. It is suggested that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes. Unfortunately, there are no DGK isozyme-specific inhibitors/activators at present. Development of these compounds is eagerly awaited for the development of novel drugs targeting DGKs.