Differential effects of ischemia and reperfusion on c-Jun N-terminal kinase isoform protein and activity.

Activation of the c-Jun N-terminal (JNK) or stress-activated protein kinases (SAPK) is associated with a wide range of disparate cellular responses to extracellular stimuli, including either induction of or protection from apoptosis. This study investigates the effect of ischemia and reperfusion on JNK isoform activities using a reversible rabbit spinal cord ischemia model. High basal JNK activity, attributed to the p46 JNK1 isoform, was expressed in the CNS of untreated rabbits. JNK activity decreased in the lumbar spinal cord of rabbits occluded for 15-60 min. During reperfusion animals occluded for 15 min recovered neurological function and JNK activity returned to normal levels. In contrast animals occluded for 60 min remained permanently paraplegic and JNK activity was half the control activity after 18 h of reperfusion. In these animals proteolytic fragments of JNK1 and JNK3 were observed and protein levels, but not activity, of JNK isoforms increased in a detergent-insoluble fraction. Two novel c-Jun (and ATF-2) kinase activities increased during reperfusion of animals occluded for 60 min. An activity designated p46(slow) was similar in M(r) to a JNK2 isoform induced in these animals. A second 30-kDa activity associated with the detergent-insoluble fraction co-migrated with a JNK3 N-terminal fragment. The results show that JNK1 is active in the normal CNS and increased activity is not associated with durations of ischemia and reperfusion that induce cell death. However, specific JNK isoform activation may participate in the cell death pathways as increased activity of novel c-Jun (ATF-2) kinase activities was observed in paraplegic animals.

Prosaptide exacerbates ischemia-induced behavioral deficits in vivo; an effect that does not involve mitogen-activated protein kinase activation.

Prosaposin is a 517 amino acid membrane component and secreted protein(5,7,9) that is proteolytically cleaved to generate the four small glycoproteins; saposins A, B, C and D.(9,13,19) Prosaposin's ability to promote neurite outgrowth(31) and to protect neurons from programmed cell death(28) in vitro, as well as to rescue neurons from ischemia and other damage in vivo(11,12,15,25) implied that prosaposin was neurotrophic/neuroprotectant.(1,7,24,31) The neurotrophic sequence of prosaposin was isolated to smaller peptide fragments termed prosaptides(15,31) within the amino terminal portion of saposin C.(1,6,8,10,17,20,21,28) The proposed use of synthetic prosaptides as peripherally administered neuroprotective and/or neurotrophic therapeutic agents has stemmed from their ability to cross the blood-brain barrier,(27) as well as their reported neurotrophic activity in vitro.(15,23,31) Few studies, however, have attempted to characterize these peptides, presumably due to their reported instability following peripheral administration.(27) With the recent design of a stable 11-mer retro-inverso prosaptide,(15,31) it has become feasible to investigate the pharmacological effects of a stable version of these peptides in the validated rabbit spinal cord ischemia model that has been used extensively in the development of therapeutics to treat ischemic stroke.(4,14,16,18) Our results show not only that prosaptide was not neurotrophic/neuroprotectant in vivo, but rather it worsened ischemia-induced behavioral deficits.

Changes in expression of the DNA repair protein complex DNA-dependent protein kinase after ischemia and reperfusion.

Reperfusion of ischemic tissue causes an immediate increase in DNA damage, including base lesions and strand breaks. Damage is reversible in surviving regions indicating that repair mechanisms are operable. DNA strand breaks are repaired by nonhomologous end joining in mammalian cells. This process requires DNA-dependent protein kinase (DNA-PK), composed of heterodimeric Ku antigen and a 460,000 Da catalytic subunit (DNA-PKcs). In this study, a rabbit spinal cord model of reversible ischemia was used to demonstrate the effect of acute CNS injury on the activity and expression of DNA-dependent protein kinase. The DNA-binding activity of Ku antigen, analyzed by an electrophoretic mobility shift assay, increased during reperfusion after a short ischemic insult (15 min of occlusion), from which the animals recover neurological function. After severe ischemic injury (60 min of occlusion) and reperfusion that results in permanent paraplegia, Ku DNA binding was reduced. Protein levels of the DNA-PK components-Ku70, Ku80, and DNA-PKcs-were monitored by immunoblotting. After 60 min of occlusion, the amount of DNA-PKcs and the enzyme poly(ADP-ribose) polymerase (PARP) decreased with the same time course during reperfusion. Concurrently 150 and 120 kDa fragments were immunostained by an anti-DNA-PKcs monoclonal antibody. This antibody was shown to cross-react with alpha-fodrin breakdown products. The 120 kDa fodrin peptide is associated with caspase-3 activation during apoptosis. Both DNA-PKcs and PARP are also substrates for caspase-3-like activities. The results are consistent with a model in which after a short ischemic insult, DNA repair proteins such as DNA-PK are activated. After severe ischemic injury, DNA damage overwhelms repair capabilities, and cell death programs are initiated.

Activation of nuclear factor-kappaB in the rabbit spinal cord following ischemia and reperfusion.

The transcription factor NF-kappaB is a ubiquitously expressed inducible regulator of a broad range of genes. Recent studies have shown that activation of NF-kappaB predominantly is associated with protecting cells from apoptosis, but in some cell models, it is associated with promoting cell death. We used a rabbit spinal cord model of reversible ischemia to determine whether NF-kappaB was activated by ischemic and reperfusion injury. DNA binding activity of NF-kappaB was analyzed by an electrophoretic mobility shift assay in animals subjected to varying durations of ischemia and reperfusion. A low level of constitutive NF-kappaB DNA binding was detected in normal lumbar spinal cord extracts. Animals subjected to a short ischemic insult of 15 min, from which they usually recover neurologic function, had a significant increase in the amount of active NF-kappaB in nuclear extracts after 18 h reperfusion. There was no change in nuclear NF-kappaB DNA binding in animals occluded for 60 min that are permanently paraplegic and exhibit extensive neuropathological damage. The amount of deoxycholate-releasable NF-kappaB sequestered in the cytosol, however, decreased after 18 h reperfusion in rabbits occluded for 60 min. This correlated with a decrease in the amount of RelA(p65) NF-kappaB subunit. The results suggest that activation of NF-kappaB after a limited ischemic injury may participate in a neuroprotective response and not in cell death.

Differences in a dinucleotide repeat polymorphism in the tau gene between Caucasian and Japanese populations: implication for progressive supranuclear palsy.

Previous studies of a tau polymorphism in Caucasian subjects with progressive supranuclear palsy (PSP) showed an over-representation of one genotype, A0/A0, versus normal control subjects. This result suggested that tau may be playing a genetic role in the progression of PSP. This study examines whether the over-representation of A0/A0 is Caucasian-specific or universal to PSP. Unfortunately, we found this dinucleotide repeat was relatively non-polymorphic in Japanese subjects. As a result, the genotypes were virtually the same, A0/A0, between Japanese PSP and control subjects. However, this outcome, albeit negative, does suggest two possible roles of the tau gene in PSP pathogenesis: (1) the role of this dinucleotide repeat in PSP may be different between Caucasian and Japanese populations or (2) this repeat may not be causal for PSP but represents a marker for other molecular genetic risk factors within or close to the tau gene on chromosome 17.

Dephosphorylation of tau during transient forebrain ischemia in the rat.

The effect of transient cerebral ischemia on phosphorylation of the microtubule-associated protein (MAP) tau was investigated using the rat four-vessel occlusion model. Phosphorylation of tau is proposed to regulate its binding to microtubules, influencing the dynamics of microtubule assembly necessary for axonal growth and neurite plasticity. In this study, tau was rapidly dephosphorylated during ischemia in the hippocampus, neocortex, and striatum. Dephosphorylation of tau was observed within 5 min of occlusion and increased after 15 min in all three brain regions, regardless of their relative vulnerability to the insult. Thus, dephosphorylation of tau is an early marker of ischemia and precedes the occlusion time required to cause extensive neuronal cell death in this model. On restoration of blood flow for a little as 15 min, tau was phosphorylated at a site(s) that causes a reduction in its electrophoretic mobility. The dephosphorylation/phosphorylation of tau may alter its distribution between axon and cell body, and affect its susceptibility to proteolysis. These changes would be expected to influence microtubule stability, possibly contributing to disruption of axonal transport, but also allowing neurite remodeling in a regenerative response.

Changes in phosphorylation of tau during ischemia and reperfusion in the rabbit spinal cord.

The microtubule-associated protein tau plays an important role in the dynamics of microtubule assembly necessary for axonal growth and neurite plasticity. Ischemia disrupts the neuronal cytoskeleton both by promoting proteolysis of its components and by affecting kinase and phosphatase activities that alter its assembly. In this study the effect of ischemia and reperfusion on the expression and phosphorylation of tau was examined in a reversible model of spinal cord ischemia in rabbits. tau was found to be dephosphorylated in response to ischemia with a time course that closely matched the production of permanent paraplegia. Dephosphorylation of tau was limited to the caudal lumbar spinal cord. In a similar manner, Ca2+/calmodulin-dependent kinase II activity was reduced only in the ischemic region. Thus, dephosphorylation of tau is an early marker of ischemia as is the rapid loss of Ca2+/calmodulin-dependent kinase II activity. tau, however, was rephosphorylated rapidly during reperfusion at site(s) that cause a reduction in its electrophoretic mobility regardless of the neurological outcome. Alterations in phosphorylation or degradation of tau may affect microtubule stability, possibly contributing to disruption of axonal transport but also facilitating neurite plasticity in a regenerative response.

Effect of cerebral ischemia on calcium/calmodulin-dependent protein kinase II activity and phosphorylation.

The effects of cerebral ischemia on calcium/calmodulin-dependent kinase II (CaM kinase II) were investigated using the rat four-vessel occlusion model. In agreement with previous results using rat or gerbil models of cerebral ischemia or a rabbit model of spinal cord ischemia, this report demonstrates that transient forebrain ischemia leads to a reduction in CaM kinase II activity within 5 min of occlusion onset. Loss of activity from the cytosol fractions of homogenates from the neocortex, striatum, and hippocampus correlated with a decrease in the amount of CaM kinase alpha and beta isoforms detected by immunoblotting. In contrast, there was an apparent increase in the amount of CaM kinase alpha and beta in the particulate fractions. The decrease in the amount of CaM kinase isoforms from the cytosol but not the particulate fractions was confirmed by autophosphorylation of CaM kinase II after denaturation and renaturation in situ of the blotted proteins. These results indicate that ischemia causes a rapid inhibition of CaM kinase II activity and a change in the partitioning of the enzyme between the cytosol and particulate fractions. CaM kinase II is a multifunctional protein kinase, and the loss of activity may play a critical role in initiating the changes leading to ischemia-induced cell death. To identify a structural basis for the decrease in enzyme activity, tryptic peptide maps of CaM kinase II phosphorylated in vitro were compared. Phosphopeptide maps of CaM kinase alpha from particulate fractions of control and ischemic samples revealed not only reduced incorporation of phosphate into the protein but also the absence of a limited number of peptides in the ischemic samples. This suggested that certain sites are inaccessible, possibly due to a conformational change, a covalent modification of CaM kinase II, or steric hindrance by an associated molecule. Verifying one of these possibilities should help to elucidate the mechanism of ischemia-induced modulation of CaM kinase II.

Inactivation and subcellular redistribution of Ca2+/calmodulin-dependent protein kinase II following spinal cord ischemia.

Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II alpha and beta subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the alpha and beta subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (< or = 25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.

Renaturation of calcium/calmodulin-dependent protein kinase activity after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to membranes.

A method is described for analyzing calcium/calmodulin-dependent protein kinase activity in crude or purified samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membrane. The blotted protein is denatured in situ with guanidine HCl and renatured in buffer containing NP-40. The membrane with bound protein is incubated with [gamma-32P]ATP and buffer containing the activators Ca2+ and calmodulin resulting in autophosphorylation of a subset of bound kinases. Two of the three major kinase activities detected in as little as 5 micrograms of crude brain or spinal cord homogenates are the alpha (M(r) = 50-52,000) and beta (M(r) = 58-62,000) isoforms of Ca2+/calmodulin-dependent protein kinase II. A third unidentified kinase of M(r) = 90-95,000 is not dependent on Ca2+ and calmodulin for activity. The membrane can be used for immunoblotting, phosphoamino acid analysis, or peptide mapping after the in situ renaturation and phosphorylation procedure. Detection of kinase activity in this assay is dependent on autophosphorylation of the enzyme. Therefore another procedure is described in which the blotted proteins are denatured and renatured in situ and assayed by measuring incorporation of phosphate into an exogenous peptide substrate specific for calcium/calmodulin-dependent protein kinase II.

Ligand-stimulated tyrosine phosphorylation of the IL-2 receptor beta chain and receptor-associated proteins.

Interleukin-2 (IL-2) stimulates the rapid phosphorylation on tyrosine of several specific cellular proteins. However, the high-affinity human IL-2 receptor, composed of an alpha (p55) and beta (p70/75) subunit, does not contain a cytoplasmic tyrosine kinase domain. In this study, we investigated the identities of the proteins phosphorylated on tyrosine in response to IL-2 stimulation to examine possible pathways of signal transduction. By the use of immunoblotting with anti-phosphotyrosine antibodies, we demonstrate that IL-2 augments tyrosine phosphorylation of the IL-2 receptor beta chain in human cell lines expressing either high-affinity (alpha/beta) receptors or only the beta chain. In IL-2-dependent mouse T cell lines, a 100,000-Da protein was phosphorylated on tyrosine in response to IL-2 and is proposed to be the mouse IL-2 receptor beta chain. Two other cellular proteins, pp55 and pp105 in human or pp55 and pp115 in mouse cell lines, were phosphorylated on tyrosine in response to IL-2 and coimmunoprecipitated with the high-affinity IL-2 receptor after chemical crosslinking of IL-2-stimulated cells. Thus, the IL-2 receptor may associate with additional subunits or with cellular proteins involved in signal transduction.

Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines.

CD45 is a family of high molecular weight leukocyte cell surface glycoproteins. Recently, two related subregions of the cytoplasmic domain of CD45 have been shown to have 30-40% amino acid identity with a human placental protein phosphotyrosine phosphatase, and CD45 isolated from human spleen was found to exhibit intrinsic protein phosphotyrosine phosphatase (EC 3.1.3.48) activity. In the present studies, we demonstrate that each of the known isoforms of murine CD45 has an equivalent basal level of protein phosphotyrosine phosphatase activity and establish that this enzymatic activity is associated with the cytoplasmic domain of the glycoprotein. Studies with three independent sets of well-characterized parental CD45+, mutant CD45-, and revertant CD45+ lymphoma cell lines indicate that loss of CD45 increases the phosphorylation of the src-related leukocyte-specific tyrosine protein kinase p56lck on tyrosine-505, a putative negative regulatory site. This suggests that CD45 may play a role in leukocyte growth regulation by altering the kinase activity of p56lck.

Changes in gene expression induced by a phorbol diester: expression of IL 2 receptor, T3, and T cell antigen receptor.

Phorbol esters cause an apparent differentiation of human T leukemic cell lines. It was shown previously that TPA induces the expression of the interleukin 2 (IL 2) receptor and the T3 complex on some T cell lines, including CCRF-CEM. We demonstrate that expression of the IL 2 receptor correlated with an induction of the 3.5 and 1.5 kb IL 2 receptor mRNA. In addition, the TPA-induced expression of the T3 polypeptides was found to be accompanied by induction of a putative T cell antigen receptor heterodimer on CEM cells. This was demonstrated by the co-precipitation of the T cell receptor with T3 from digitonin-solubilized cells. The cells expressed high levels of T3 delta- and T cell receptor beta-chain mRNA in the absence of TPA. The effect of TPA was to cause a rapid accumulation of T cell receptor alpha-chain mRNA. This suggested that the alpha-chain gene was rearranged before TPA induction and that expression of the T cell receptor/T3 complex on the cell surface was regulated by the level of alpha-chain expression. It was also shown that cloned sublines of CEM cells which expressed different T cell antigen phenotypes differed in their response to TPA.

Evidence for two extracellular domains in the human interleukin-2 receptor: localization of IL-2 binding.

The human interleukin-2 (IL-2) receptor was quantitatively cleaved into two large disulfide-bonded fragments by either trypsin or endoproteinase lys-C (endo lys-C). The smaller fragment contains both N-linked oligosaccharides found in the intact receptor and is derived from the amino terminus of the molecule. The larger proteolytic fragment was metabolically labeled with 32PO4 and represents the carboxy terminus. The predicted cleavage sites of both enzymes lie in the region of the molecule encoded by exon 3. This pattern of limited proteolysis provides biochemical evidence that the extracellular region of the receptor is organized into two domains. This supports a structural model of the receptor in which the regions of internal homology encoded by exons 2 and 4 form independent disulfide-bonded domains connected by a hydrophilic segment. To determine the role of these domains in IL-2 binding, [125I]IL-2 was chemically cross-linked to the proteolytically cleaved receptor on the cell surface. The 125I-labeled complex obtained displayed N-linked oligosaccharides and had an Mr consistent with one molecule of IL-2 cross-linked to the smaller proteolytic fragment of the receptor. Thus, the amino-terminal domain of the IL-2 receptor appears to form an integral part of the IL-2 binding site.

Identification of lymphocyte integral membrane proteins as substrates for protein kinase C. Phosphorylation of the interleukin-2 receptor, class I HLA antigens, and T200 glycoprotein.

The interleukin-2 (IL-2) receptor, the leukocyte-specific membrane glycoprotein, T200, and the class I major histocompatibility antigens (HLA) have been identified as substrates for protein kinase C in vitro. IL-2 receptors on normal human T lymphocytes and the leukemic cell line, HUT102B2, are rapidly phosphorylated in vivo in response to the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Tryptic peptide analysis showed that the in vitro and in vivo 32P-labeled IL-2 receptors were phosphorylated on the same sites. A synthetic peptide corresponding to the carboxyl-terminal cytoplasmic tail of the IL-2 receptor was shown to be phosphorylated in vitro by protein kinase C. Tryptic digestion of the peptide generated the same 32P-labeled species as those found for the IL-2 receptor. From these studies, it was concluded that Ser-247 is the major site of phosphorylation in the IL-2 receptor and that Thr-250 is a minor site. These results also provide direct evidence that the in vivo phosphorylation of the IL-2 receptor stimulated by TPA is catalyzed by protein kinase C. The sites phosphorylated in the HLA antigens in vitro by protein kinase C or in vivo after TPA stimulation were also localized to the carboxyl-terminal cytoplasmic domain of the heavy chain by limited proteolysis.

Structure and function of transferrin receptors and their relationship to cell growth.

The transferrin receptor binds the major serum iron-transport protein, transferrin, and mediates cellular iron uptake. The receptor is a major immunodominant cell surface glycoprotein of cultured cells and its expression on the cell surface is co-ordinately regulated with cell growth. Recent structural and functional studies of the transferrin receptor are reviewed. The properties of monoclonal antibodies against the transferrin receptor that inhibit transferrin-mediated iron uptake are described. Studies with these antibodies establish that the transferrin receptor plays an important role in cell growth and suggest that monoclonal antibodies that interfere with the function of growth-related receptors may be useful in regulating tumour cell growth.

Induction of expression and phosphorylation of the human interleukin 2 receptor by a phorbol diester.

The receptor for interleukin 2 (IL 2) is induced on normal mature T cells activated by antigen or mitogen. Receptor-negative human T leukemic cell lines were induced to express the IL 2 receptor after incubation with 12-O-tetradecanoyl phorbol 13-acetate. The Mr of the IL 2 receptor expressed on the surface of four different T cell lines and normal peripheral blood lymphocytes varied. The source of the heterogeneity appeared to be due to differences in post-translational processing of the receptor. The precursor of the IL 2 receptor isolated from tunicamycin-treated HUT102B2 or 12-O-tetradecanoyl phorbol 13-acetate-induced CCRF-CEM cells had the same Mr and isoelectric point. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate also induced the rapid phosphorylation of serine and threonine residues on the IL 2 receptor from HUT102B2 cells and activated peripheral blood lymphocytes. This phosphorylation may be mediated by the Ca2+ and phospholipid-dependent protein kinase C.

Identification of three class II antigens, DR7, MB2, and MT3, from a homozygous human cell line.

The class II antigens from a homozygous DR7 human B cell line were isolated by immunoprecipitation with human alloantisera or monoclonal antibodies. Three class II light chains and three class II heavy chains were identified. The heavy and light chain subunits of the DR7 antigen were distinct from those of the MB2 antigen as assessed by two-dimensional gel electrophoresis. A third light chain in addition to the DR7 and MB2 subunits was precipitated by some alloantisera containing MT3 antibodies. The monoclonal antibodies examined immunoprecipitated one or more of the three class II antigens. The MB2 antigen appeared to be the same as the major antigen recognized by L227 and the DS7 antigen precipitated by SG171.

Analysis of the oligosaccharides on the HLA-DR and DC1 B cell antigens.

The types of N-linked oligosaccharides on the HLA-DR and DC1 antigens were determined by their sensitivity to endoglycosidase H or endoglycosidase D. The DR and DC1 heavy chains each have two carbohydrate moieties, one high-mannose and one complex-type glycan. The DR and DC1 light chains have one complex-type oligosaccharide. During the biosynthesis of the DR antigens, the oligosaccharides on both subunits are initially high-mannose-type glycans, as has been found for other membrane glycoproteins. The light chain oligosaccharide and one of the two heavy chain carbohydrates are later processed to complex-type glycans. Inhibition of N-linked glycosylation with tunicamycin does not inhibit chain association of the DR and DC1 subunits, transport to the cell surface, or expression of the alloantigenic determinants.