Substitution of the autophosphorylation site Thr516 with a negatively charged residue confers constitutive activity to mouse 3-phosphoinositide-dependent protein kinase-1 in cells

3-Phosphoinositide-dependent protein kinase-1 (PDK-1)is a serine/threonine kinase that has been found to phosphorylate and activate several members of the AGC protein kinase family including protein kinase B (Akt), p70 S6 kinase, and protein kinase Czeta. However, the mechanism(s) by which PDK-1 is regulated remains unclear. Here we show that mouse PDK-1 (mPDK-1) undergoes autophosphorylation in vitro on both serine and threonine residues. In addition, we have identified Ser(399) and Thr(516) as the major mPDK-1 autophosphorylation sites in vitro. Furthermore, we have found that these two residues, as well as Ser(244) in the activation loop, are phosphorylated in cells and demonstrated that Ser(244) is a major in vivo phosphorylation site. Abolishment of phosphorylation at Ser(244), but not at Ser(399) or Thr(516), led to a significant decrease of mPDK-1 autophosphorylation and kinase activity in vitro, indicating that autophosphorylation at Ser(399) or Thr(516) is not essential for mPDK-1 autokinase activity. However, overexpression of mPDK-1(T516E), but not of mPDK-1(S244E) or mPDK-1(S399D), in Chinese hamster ovary and HEK293 cells was sufficient to induce Akt phosphorylation at Thr(308) to a level similar to that of insulin stimulation. Furthermore, this increase in phosphorylation was independent of the Pleckstrin homology domain of Akt. Taken together, our results suggest that mPDK-1 undergoes autophosphorylation at multiple sites and that this phosphorylation may be essential for PDK-1 to interact with and phosphorylate its downstream substrates in vivo.     

Detection of viral DNA and immune responses to the human herpesvirus 6 101-kilodalton virion protein in patients with multiple sclerosis and in controls

Human herpesvirus 6 (HHV-6), a latent lymphotropic and neurotropic virus, has been suspected as an etiologic agent in multiple sclerosis (MS). The study was undertaken to correlate virologic evidence for HHV-6 activity with the state of host immunity to HHV-6 in MS patients and control subjects. The study revealed that cell-free DNA of HHV-6 was detected more frequently in both serum and cerebrospinal fluid of MS patients than in those of control subjects. T cells recognizing the recombinant 101-kDa protein (101K) corresponding to the major immunoreactive region unique to HHV-6 occurred at significantly lower precursor frequency in MS patients than in control subjects. The resulting HHV-6-specific T-cell lines obtained from MS patients exhibited skewed cytokine profiles characterized by the inability to produce interleukin-4 (IL-4) and IL-10. The decreased T-cell responses to HHV-6 and the altered cytokine profile were consistent with significantly declined serum immunoglobulin G (IgG) titers for HHV-6 of MS patients compared to those of control subjects. In contrast, elevated serum IgM titers for HHV-6 were detected in the majority of MS patients, which may reflect frequent exposure of B cells to HHV-6. The findings suggest that the decreased immune responses to HHV-6 may be responsible for ineffective clearance of HHV-6 in MS patients.     

Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta

3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.     

The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer's and Parkinson's diseases

Alzheimer's (AD) and Parkinson's diseases (PD) are late-onset neurodegenerative diseases that have tremendous impact on the lives of affected individuals, their families, and society as a whole. Remarkable efforts are being made to elucidate the dominant factors that result in the pathogenesis of these disorders. Extensive postmortem studies suggest that oxidative/nitrative stresses are prominent features of these diseases, and several animal models support this notion. Furthermore, it is likely that protein modifications resulting from oxidative/nitrative damage contribute to the formation of intracytoplasmic inclusions characteristic of each disease. The frequent presentation of both AD and PD in individuals and the co-occurrence of inclusions characteristic of AD and PD in several other neurodegenerative diseases suggests the involvement of a common underlying aberrant process. It can be surmised that oxidative/nitrative stress, which is cooperatively influenced by environmental factors, genetic predisposition, and senescence, may be a link between these disorders.     

Use of online-dual-column extraction in conjunction with chiral liquid chromatography tandem mass spectrometry for determination of terbutaline enantiomers in human plasma

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a beta2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0-200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94-106%.     

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity

Abstract. The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1–24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5¢-amino-5¢-deoxythymidine (5¢-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.