Anti-Tribbles Pseudokinase 2 (TRIB2)-Immunization Modulates Hypocretin/Orexin Neuronal Functions.

Study Objectives: Recent findings showed that 16%-26% of narcolepsy patients were positive for anti-tribbles pseudokinase 2 (TRIB2) antibody, and the intracerebroventricular administration of immunoglobulin-G purified from anti-TRIB2 positive narcolepsy patients caused hypocretin/orexin neuron loss. We investigated the pathophysiological role of TRIB2 antibody using TRIB2-immunized rats and hypocretin/ataxin-3 transgenic (ataxin-3) mice. Methods: Plasma, cerebrospinal fluid (CSF), and hypothalamic tissues from TRIB2-immunized rats were collected. Anti-TRIB2 titers, hypocretin contents, mRNA expressions, the cell count of hypocretin neurons, and immunoreactivity of anti-TRIB2 antibodies on hypocretin neurons were investigated. The plasma from ataxin-3 mice was also used to determine the anti-TRIB2 antibody titer changes following the loss of hypocretin neurons. Results: TRIB2 antibody titers increased in the plasma and CSF of TRIB2-immunized rats. The hypothalamic tissue immunostained with the sera from TRIB2-immunized rats revealed positive signals in the cytoplasm of hypcretin neurons. While no changes were found regarding hypothalamic hypocretin contents or cell counts, but there were significant decreases of the hypocretin mRNA level and release into the CSF. The plasma from over 26-week-old ataxin-3 mice, at the advanced stage of hypocretin cell destruction, showed positive reactions against TRIB2 antigen, and positive plasma also reacted with murine hypothalamic hypocretin neurons. Conclusions: Our results suggest that the general activation of the immune system modulates the functions of hypocretin neurons. The absence of a change in hypocretin cell populations suggested that factors other than anti-TRIB2 antibody play a part in the loss of hypocretin neurons in narcolepsy. The increased anti-TRIB2 antibody after the destruction of hypocretin neurons suggest that anti-TRIB2 antibody in narcolepsy patients is the consequence rather than the inciting cause of hypocretin cell destruction.

A new approach for the detection of multiple protein kinases using monoclonal antibodies directed to the highly conserved region of protein kinases.

To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.

Detection of anti-elongation factor 2 kinase (calmodulin-dependent protein kinase III) antibodies in patients with systemic lupus erythematosus.

Elongation factor 2 kinase (eEF-2K), also known as calmodulin-dependent protein kinase III, is a member of the calmodulin-mediated signaling pathway that links activation of cell surface receptors to cell division. The activity of eEF-2K is increased in many human cancers and may be a valid target for anti-cancer treatment. It is one of the unconventional eukaryotic protein kinases with respect to its structural domains in comparison to other members of the serine/threonine protein kinase superfamily. eEF-2K is highly conserved in nature. For example, the amino acid sequence of human eEF-2K is 90% identical to mouse and rat eEF-2Ks and 40% identical to that of the C. elegans enzyme. Therefore it has been difficult to generate high-titer and high-specificity antibodies to the human enzyme by traditional techniques. Patients with systemic lupus erythematosus (SLE) produce auto-antibodies to a variety of cellular proteins, including members of the protein translation apparatus. Hence, we developed an ELISA assay that could detect anti-eEF2K antibodies from sera of SLE patients using purified eEF-2K as an antigen. We screened 117 sera from SLE patients. High-titer anti-eEF-2K antibodies were detected in 72 subjects. One of the high-titer sera was used for further characterization. The auto-antibody recognized eEF-2K on immunoblots and immunoprecipitated the kinase with intact enzyme activity. In conclusion, anti-eEF-2K antibodies are found in sera of SLE patients and are useful tools to study the role of this highly conserved enzyme.

Mutated mitogen-activated protein kinase: a tumor rejection antigen of mouse sarcoma.

The molecular basis of the polymorphic tumor rejection antigens of chemically induced sarcomas of inbred mice remains a mystery, despite the discovery of these antigens over 40 years ago and their critical importance to the foundation of tumor immunology. In an analysis of a panel of BALB/c 3-methylcholanthrene-induced tumors, we identified one tumor, CMS5, that elicited a strong cytotoxic T cell response with exquisite specificity for CMS5. A stable cloned line of T cells with this specificity (C18) was used to screen a CMS5 cDNA expression library. The gene encoding the C18-defined antigen was identified as a mutated form of a mouse mitogen-activated protein kinase, ERK2, and a peptide incorporating the resulting amino acid substitution (lysine to glutamine) was efficiently recognized by C18. Vaccination with this peptide elicited specific resistance to CMS5 challenge. Extensive efforts to isolate antigen-loss variants of CMS5 were unsuccessful, suggesting that the mutated mitogen-activated protein kinase is essential for maintenance of the malignant phenotype.

A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3.

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 was shown to phosphorylate and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 was also shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 were strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells resulted in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.

Alpha calcium/calmodulin-dependent protein kinase II immunoreactivity in corticospinal neurons: combination of axonal transport method and immunofluorescence.

A combination of either retrograde or anterograde fluorescent tracer and immunofluorescence histochemistry using the monoclonal antibody specific for the alpha isoform of calcium/calmodulin-dependent protein kinase II (CaM kinase II alpha) was employed to test whether CaM kinase II alpha is expressed in somata of corticospinal neurons and their axons over their whole course. After the injection of carbocyanine dye DiI into the hindlimb area of the primary motor cortex of the rat, corticospinal axons and their terminal arbors were anterogradely labeled: DiI-labeled corticospinal fibers proceeded caudally in the ipsilateral internal capsule, cerebral peduncle and medullary pyramid, crossed at the pyramidal decussation and descended in the ventralmost area of the contralateral dorsal funiculus of the spinal cord. These DiI-labeled corticospinal axons expressed strong CaM kinase II alpha immunoreactivity along their course. However, their terminal arbors within the gray matter of the lumbar cord were very weakly immunostained. With the injection of Fast Blue into the lumbar enlargement of the rat, somata of corticospinal neurons in layer V of the motor cortex were retrogradely labeled. The subsequent immunofluorescent histochemistry revealed that more than 80% of Fast Blue-labeled corticospinal neurons were immunostained with CaM kinase II alpha antibody. The present immunohistochemical study demonstrated that CaM kinase II alpha is strongly expressed in both somata and axons of a majority of corticospinal neurons, although we could not detect this enzyme in the corticospinal terminals in the spinal target areas.

The cDNA sequence and characterization of the Ca2+/calmodulin-dependent protein kinase-Gr from human brain and thymus.

We have isolated and sequenced cDNAs encoding Ca2+/calmodulin-dependent protein kinase type Gr (CaM-K-Gr, also called CaM-K-IV) from human brain and thymus. The sequence of the protein coding region of the cDNA is identical in both brain and thymus, although Northern hybridization analysis shows variation of the mRNA transcripts in these tissues. The sequence predicts a protein of M(r) 51,897 that is 83.7% identical and shows 89.2% similarity with the rat homologue. The deduced human CaM-K-Gr is identical to the rat and mouse proteins in the portion of the enzyme involved in ATP binding, the catalytic domain and Ca2+/calmodulin-binding domain; however, the N terminus of the human kinase, which may comprise a second regulatory domain [McDonald et al., J. Biol. Chem. 268 (1993) 10054-10059], contains a 4-amino-acid (aa) insertion relative to the rodent enzymes. Additionally, the C-terminal association domain shows only 45.2 and 41.6% identity with the rat and mouse proteins, respectively, suggesting that this domain is not constrained by stringent structural and functional requirements. Based on the predicted aa sequence of the human kinase, we produced polyclonal antisera against a C-terminal peptide that recognizes two forms of CaM-K-Gr in human T-cell lymphoma and neuroblastoma cell lines. The human antiserum cross-reacts with the rat and mouse proteins and immunoprecipitates the active kinase.

Immunohistochemical detection of calcium/calmodulin-dependent protein kinase II in the spinal cord of the rat and monkey with special reference to the corticospinal tract.

Calcium/calmodulin-dependent protein kinase II is a prominent enzyme in the mammalian brain that phosphorylates a variety of substrate proteins. In the present study, monoclonal antibodies that specifically recognize either the alpha or the beta isoforms of this enzyme were used to determine the distribution of these isoforms within the rat and monkey spinal cord. In the rat, the corticospinal tract consists of two components: the dorsal corticospinal tract, which occupies the ventralmost aspect of the dorsal funiculus; and the ventral corticospinal tract, which occupies an area adjacent to the ventral median fissure. Both dorsal and ventral corticospinal tract fibers were strongly immunopositive for the alpha-antibody. Unilateral ablation of the sensorimotor cortex of the rat eliminated the alpha-immunoreactive staining in the contralateral dorsal corticospinal tract. The neuropil in the superficial laminae of the dorsal horn (Rexed's laminae I and II) was densely stained with the alpha-antibody, whereas the neuropil in laminae IV-X was immunonegative. Dense alpha-immunopositive neurons were also distributed in the head of the dorsal horn (laminae I-IV). In contrast to the strong alpha-immunoreactivity seen in the dorsal corticospinal tract fibers, only very weak beta-immunoreactivity was observed in this tract. Moderate beta-immunoreactive products were distributed homogenously throughout the neuropil of the gray matter, although the neuropil of the superficial laminae of the dorsal horn (laminae I and II) was stained more strongly than the other regions of the gray matter (laminae III-X). Neuronal components in all laminae were immunopositive for the beta-antibody. Thus, motoneurons in the ventral horn, which were immunonegative for the alpha-antibody, were immunopositive for the beta-antibody. This selective distribution pattern of immunoreactivity of alpha- and beta-antibodies in the rat was also present in the monkey spinal cord, although the alpha-immunopositive corticospinal tract fibers in the monkey descended in the lateral funiculus as the lateral corticospinal tract instead of passing through the dorsal funiculus, as is the case in the rat. The differential distribution of immunoreactivity in the spinal cord suggests that these two isoforms of calcium/calmodulin-dependent protein kinase II may have different functional roles in the spinal cord.