Non-thromboembolic risk in systemic lupus erythematosus associated with antiphospholipid syndrome.

OBJECTIVES: We investigated the impact of secondary antiphospholipid syndrome (APS) and antiphospholipid antibody (aPL) positivity on the non-thromboembolic clinical manifestations of systemic lupus erythematosus (SLE). METHODS: In total, 224 patients with SLE were studied, of whom 105 were aPL-positive; 52 fulfilled the criteria for APS. SLE- and APS-related clinical and laboratory features were assesed: SLE patients with aPL or APS were compared with those without these features. RESULTS: Not only thromboembolic events, but also Coombs-positive haemolytic anaemia, thrombocytopenia and endocarditis occurred significantly more frequently in the aPL-positive than in the aPL-negative patients. In the APS + SLE subgroup, several non-thromboembolic symptoms occurred more often than in the absence of APS: pleuritis, interstitial lung disease, myocarditis, nephritis and organic brain syndrome. The mean number of major organ manifestations (1.2 vs. 0.5) and the overall number of organ manifestations (8.1 vs. 6.9) were higher in the APS + SLE patients than in those without APS (p < 0.05). The APS + SLE subgroup more frequently required intensive immunosuppressive treatment than did the APS-negative patients (p < 0.05). CONCLUSIONS: SLE patients with aPL positivity or secondary APS also have a higher risk to develop non-thromboembolic disease manifestations in addition to the aPL-related symptoms, and are predisposed to more severe SLE manifestations.

Sleep duration and timing in obsessive-compulsive disorder (OCD): evidence for circadian phase delay.

OBJECTIVES: To investigate potential delays in endogenous melatonin in individuals with obsessive-compulsive disorder (OCD). METHODS: First, data are presented for 15 individuals with OCD and matched healthy controls. Next, nine additional participants with OCD who did not have matched controls were added, resulting in a sample of 24 individuals with OCD. All participants were assessed for sleep and circadian rhythm disturbance. Dim light melatonin onset (DLMO) was derived from salivary melatonin and was used in conjunction with sleep diaries, interview measures, and questionnaires. A subset of the OCD group (n = 16) also used actigraphy. RESULTS: In sum, 42% percent (10/24) of the patients with OCD met the criteria for delayed sleep-wake phase disorder (DSWPD) in comparison to 0% in the control sample. DLMO was significantly later in individuals with OCD compared to controls. DLMO and bedtime were not significantly associated with the severity of obsessive-compulsive symptoms or negative affect. CONCLUSIONS: Replication of the findings presented herein, particularly the DLMO results, is warranted. Further, there are now three studies showing that nearly ½ of individuals with OCD meet criteria for a DSWPD. Future studies can explore the mechanisms underlying these connections and the implications of this comorbidity. These findings may increase our understanding of OCD and inform future interventions.

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2.

BACKGROUND: Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown. RESULTS: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBalpha, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBalpha to one that phosphorylates both Thr308 and Ser473 of PKBalpha in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBalpha in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies. CONCLUSIONS: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase.

BACKGROUND: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2). RESULTS: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. CONCLUSIONS: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

Rice Phytochrome Is Biologically Active in Transgenic Tobacco.

To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.

Plants ectopically expressing the iron-binding protein, ferritin, are tolerant to oxidative damage and pathogens.

Transgenic tobacco plants that synthesize alfalfa ferritin in vegetative tissues--either in its processed form in chloroplasts or in the cytoplasmic nonprocessed form--retained photosynthetic function upon free radical toxicity generated by iron excess or paraquat treatment. Progeny of transgenic plants accumulating ferritin in their leaves exhibited tolerance to necrotic damage caused by viral (tobacco necrosis virus) and fungal (Alternaria alternata, Botrytis cinerea) infections. These transformants exhibited normal photosynthetic function and chlorophyll content under greenhouse conditions. We propose that by sequestering intracellular iron involved in generation of the very reactive hydroxyl radicals through a Fenton reaction, ferritin protects plant cells from oxidative damage induced by a wide range of stresses.

A cis-acting region required for the regulated expression of grg-1, a Neurospora glucose-repressible gene. Two regulatory sites (CRE and NRS) are required to repress grg-1 expression.

Grg-1 is a Neurospora gene which was identified as being a highly expressed glucose-repressible gene. A cis-acting regulatory region required for the regulated expression of grg-1 has been characterized. The regulatory region is found between 440 and 500 nucleotides upstream of the first major grg-1 start of transcription site and contains two distinct cis-acting regulatory elements. The upstream element consists of the sequence GTGACGTCAC, which is identical to the previously identified CRE (Cyclic AMP-Responsive Element). The second element is a newly defined cis-acting regulatory site. The element has the sequence TTGCTAGCAA and has been named NRS (Neurospora Repressor Site). DNA binding proteins can be shown to bind to both of these cis-acting regulatory elements. Experiments in which these sites were deleted demonstrate that both cis-acting regulatory elements are required to turn off the in vitro expression of the grg-1 gene under conditions of glucose sufficiency.

The systemic administration of gamma interferon inhibits collagen synthesis and acute inflammation in a murine skin wounding model.

The ability of gamma interferon (IFN-gamma) to affect cutaneous collagen synthesis in vivo was examined in a murine wounding model. Reproducible areas of full-thickness skin necrosis were produced by argon laser radiation. Mice received recombinant murine IFN-gamma (rMuIFN-gamma) (8.7 X 10(3) units/hr) over 14 d via osmotic pumps implanted subcutaneously or intraperitoneally. At 14 and 21 d after wounding, there was less fibrous tissue in healing scars of treated animals as determined by light and transmission electron microscopy. Associated with the decrease in connective tissue was an increase in the acid mucopolysaccharide content of healing scars, which was largely hyaluronate. Quantitative image analysis of electron micrographs confirmed that less collagen was present in healing scars of animals receiving rMuIFN-gamma. The mean cross-sectional area of collagen fibers was smaller in specimens from treated mice, but no difference was seen in the size of collagen fibrils. The time required to obtain full skin closure was also delayed 23%-27% in treated animals. Using this injury model, we also found that rMuIFN-gamma significantly reduced the degree of perilesional erythema surrounding the laser injury sites and, in the first 6 d after wounding, the degree of polymorphonuclear infiltrate present histologically at lesional sites. Indeed, rMuIFN-gamma also decreased the cutaneous accumulation of neutrophils induced by known proinflammatory mediators, such as interleukin 1 and activated serum. Thus, systemically administered IFN-gamma not only down-regulates collagen synthesis in the skin but also modulates in a previously unrecognized manner: neutrophil accumulation at sites of tissue injury in vivo.

Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain.

A plant homologue of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3-phosphoinositide-dependent protein kinase-1. Like the mammalian 3-phosphoinositide-dependent protein kinase-1, Arabidopsis 3-phosphoinositide-dependent protein kinase-1 and rice 3-phosphoinositide-dependent protein kinase-1 possess a kinase domain at N-termini and a pleckstrin homology domain at their C-termini. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3-phosphoinositide-dependent protein kinase-1 homologues. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is able to activate human protein kinase B alpha (PKB/AKT) in the presence of PtdIns(3,4,5)P3. Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3-phosphoinositides.

Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase.

The catalytic domain of serum- and glucocorticoid-induced protein kinase (SGK) is 54% identical with protein kinase B (PKB) and, like PKB, is activated in vitro by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and in vivo in response to signals that activate phosphatidylinositol (PI) 3-kinase. Here we identify two novel isoforms of SGK, termed SGK2 and SGK3, whose catalytic domains share 80% amino acid sequence identity with each other and with SGK (renamed SGK1). Like SGK1, the mRNA encoding SGK3 is expressed in all tissues examined, but SGK2 mRNA is only present at significant levels in liver, kidney and pancreas and, at lower levels, in the brain. The levels of SGK2 mRNA in H4IIE cells and SGK3 mRNA in Rat2 fibroblasts are not increased by stimulation with serum or dexamethasone, whereas the level of SGK1 mRNA is increased greatly. SGK2 and SGK3 are activated in vitro by PDK1, albeit more slowly than SGK1, and their activation is accompanied by the phosphorylation of Thr(193) and Thr(253) respectively, the residues equivalent to the Thr in the 'activation loop' of PKB that is targeted by PDK1. The PDK1-catalysed phosphorylation and activation of SGK2 and SGK3, like SGK1, is greatly potentiated by mutating Ser(356) and Ser(419) respectively to Asp, these residues being equivalent to the C-terminal phosphorylation site of PKB. Like SGK1, SGK2 and SGK3 are activated 5-fold via a phosphorylation mechanism when cells are exposed to H(2)O(2) but, in contrast with SGK1, activation is only suppressed partially by inhibitors of PI 3-kinase. SGK2 and SGK3 are activated to a smaller extent by insulin-like growth factor-1 (2-fold) than SGK1 (5-fold). Like PKB and SGK1, SGK2 and SGK3 preferentially phosphorylate Ser and Thr residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr motifs.

Dominant expression of a gene amplification-related herbicide resistance in medicago cell hybrids.

Protoplasts from phosphinotricin resistant M. sativa and M. varia cell lines carrying an amplified glutamine synthethase gene were fused with leaf protoplasts of kanamycin resistant M. varia transformants. The dominant nature of both PPT and kanamycin resistant traits was shown by the double resistant phenotype of the intra- and interspecific cell hybrids obtained. The presence of amplified GS gene in the hybrid genomes and the expression of chimeric neomycin phosphotransferase II gene was detected. The highly embryogenic character of the M. varia parent was not expressed after cell fusion. All hybrid cell lines with the double resistant phenotype showed non-morphogenic growth similarly to the PPT resistant parent. The possible role of GS gene amplification and other factors in the dominant behaviour of unorganized cell growth in alfalfa somatic hybrids is discussed.

Transformation of Medicago by Agrobacterium mediated gene transfer.

Shoot segments of Medicago varia genotype A2 were co-cultivated with Agrobacterium tumefaciens strain bo42 carrying pGA471, a plasmid coding for the kanamycin resistant determinant as transferable positive selection marker in plant cells (An et al., 1985). Resistant plants were regenerated at high frequency from green calli developed on inoculated stem cuttings under kanamycin selection. DNA-DNA hybridization analysis showed the presence of the structural gene of the kanamycin resistant determinant in total DNA isolated from several independent transformants. All data presented clearly demonstrate the transfer, stable maintenance and functional expression of the kanamycin resistance marker in Medicago varia cells which retain their morphogenic property.

Rice type I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings.

We have examined the biological activity of rice type I phytochrome (PI) in transgenic tobacco seedlings. The progeny of four independent transformants that expressed the rice PI gene segregated 3:1 for shorter hypocotyl length under dim white light (0.04 W/m2). By contrast, this phenotype was not observed either in the dark or under white light at higher intensity (6.0 W/m2). This suggests that the phenotype is dependent not only on light but also on light intensity. The increased light sensitivity cosegregated with the kanamycin-resistance marker as well as with the rice PI polypeptides, indicating that this phenotype is directly related to the expression of the transgene. The transgenic plants showing short hypocotyls exhibited a reduced growth rate throughout the elongation period, and the resulting shorter hypocotyl length was attributable to shorter epidermal cell length but not to reduced cell number. Furthermore, successive pulse irradiations with red light elicited short hypocotyls similar to those obtained under dim white light, and the effect was reversed by immediate far-red light treatment, providing a direct indication that the phenotype is caused by biologically active rice PI. Therefore, the far-red-absorbing form of the introduced rice PI appears to regulate the hypocotyl length of the transgenic tobacco plants through endogenous signal-transduction pathways. This assay system will be a powerful tool for testing the biological activity of introduced phytochrome molecules.

Crystal structure of the phosphatidylinositol 3,4-bisphosphate-binding pleckstrin homology (PH) domain of tandem PH-domain-containing protein 1 (TAPP1): molecular basis of lipid specificity.

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and its immediate breakdown product PtdIns(3,4)P(2) function as second messengers in growth factor- and insulin-induced signalling pathways. One of the ways that these 3-phosphoinositides are known to regulate downstream signalling events is by attracting proteins that possess specific PtdIns-binding pleckstrin homology (PH) domains to the plasma membrane. Many of these proteins, such as protein kinase B, phosphoinositide-dependent kinase 1 and the dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1) interact with both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) with similar affinity. Recently, a new PH-domain-containing protein, termed tandem PH-domain-containing protein (TAPP) 1, was described which is the first protein reported to bind PtdIns(3,4)P(2) specifically. Here we describe the crystal structure of the PtdIns(3,4)P(2)-binding PH domain of TAPP1 at 1.4 A (1 A=0.1 nm) resolution in complex with an ordered citrate molecule. The structure is similar to the known structure of the PH domain of DAPP1 around the D-3 and D-4 inositol-phosphate-binding sites. However, a glycine residue adjacent to the D-5 inositol-phosphate-binding site in DAPP1 is substituted for a larger alanine residue in TAPP1, which also induces a conformational change in the neighbouring residues. We show that mutation of this glycine to alanine in DAPP1 converts DAPP1 into a TAPP1-like PH domain that only interacts with PtdIns(3,4)P(2), whereas the alanine to glycine mutation in TAPP1 permits the TAPP1 PH domain to interact with PtdIns(3,4,5)P(3).

The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB.

PKB/Akt, S6K1 and SGK are related protein kinases activated in a PI 3-kinase-dependent manner in response to insulin/growth factors signalling. Activation entails phosphorylation of these kinases at two residues, the T-loop and the hydrophobic motif. PDK1 activates S6K, SGK and PKB isoforms by phosphorylating these kinases at their T-loop. We demonstrate that a pocket in the kinase domain of PDK1, termed the 'PIF-binding pocket', plays a key role in mediating the interaction and phosphorylation of S6K1 and SGK1 at their T-loop motif by PDK1. Our data indicate that prior phosphorylation of S6K1 and SGK1 at their hydrophobic motif promotes their interaction with the PIF-binding pocket of PDK1 and their T-loop phosphorylation. Thus, the hydrophobic motif phosphorylation of S6K and SGK converts them into substrates that can be activated by PDK1. In contrast, the PIF-binding pocket of PDK1 is not required for the phosphorylation of PKBalpha by PDK1. The PIF-binding pocket represents a substrate recognition site on a protein kinase that is only required for the phosphorylation of a subset of its physiological substrates.

Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB.

We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.

Activation of protein kinase B beta and gamma isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B alpha.

The regulatory and catalytic properties of the three mammalian isoforms of protein kinase B (PKB) have been compared. All three isoforms (PKBalpha, PKBbeta and PKBgamma) were phosphorylated at similar rates and activated to similar extents by 3-phosphoinositide-dependent protein kinase-1 (PDK1). Phosphorylation and activation of each enzyme required the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, as well as PDK1. The activation of PKBbeta and PKBgamma by PDK1 was accompanied by the phosphorylation of the residues equivalent to Thr308 in PKBalpha, namely Thr309 (PKBbeta) and Thr305 (PKBgamma). PKBgamma which had been activated by PDK1 possessed a substrate specificity identical with that of PKBalpha and PKBbeta towards a range of peptides. The activation of PKBgamma and its phosphorylation at Thr305 was triggered by insulin-like growth factor-1 in 293 cells. Stimulation of rat adipocytes or rat hepatocytes with insulin induced the activation of PKBalpha and PKBbeta with similar kinetics. After stimulation of adipocytes, the activity of PKBbeta was twice that of PKBalpha, but in hepatocytes PKBalpha activity was four-fold higher than PKBbeta. Insulin induced the activation of PKBalpha in rat skeletal muscle in vivo, with little activation of PKBbeta. Insulin did not induce PKBgamma activity in adipocytes, hepatocytes or skeletal muscle, but PKBgamma was the major isoform activated by insulin in rat L6 myotubes (a skeletal-muscle cell line).

Evidence for the presence of an N-acetyllactosamine-type chain in epiglycanin.

Mannose-labeled epiglycanin was prepared by incubation of TA3-Ha ascites cells with [2-3H]mannose, removal of the epiglycanin by incubation of viable cells with L-1-p-tosylamino-2-phenylethyl chloromethyl ketone-trypsin, and isolation of the large epiglycanin glycopeptides by gel filtration. Purification of epiglycanin glycopeptides was performed by wheat germ agglutinin affinity chromatography. Extensive incubation of epiglycanin with Pronase, followed by passage through a calibrated column of Bio-Gel P-4 (Column P-4), gave three fractions. The fraction of lowest apparent molecular weight, about 5000, upon incubation with a purified extract from F. meningosepticum containing an N-glycosyl hydrolase and an endo-N-acetyl-beta-D-glucosaminidase (T.H. Plummer et. al. (1984) J. Biol. Chem. 259, 10700-10704) and passage through Column P-4 gave a peak of radioactivity at apparent Mr 3000. Incubation of nonlabeled epiglycanin under similar conditions with the same enzyme preparation followed by passage through Column P-4, gave two peaks, based upon total mannose content. One of these, partially deglycosylated epiglycanin, was present in the void volume. Its composition indicated that approximately 80% of the mannose content of epiglycanin had been removed by the enzyme treatment, whereas no change was noted in the proportion of the other carbohydrate components. The effluent volume of the second peak coincided precisely with the peak obtained from the Pronase-cleaved fraction. Its composition and apparent Mr were consistent with those of an N-lactosamine-type chain with four antennae, Man3Gal4GlcNAc5NeuAc2-3.

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA.

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP- and substrate-binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF-binding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.

A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Czeta (PKCzeta ) and PKC-related kinase 2 by PDK1.

Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKCzeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKCzeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKCzeta and PKCiota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKCzeta, full-length PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in vivo. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKCzeta. These findings indicate that the hydrophobic motif of PRK2 and PKCzeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.