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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., ((c) 2002 Elsevier Science (USA).)

We have studied the effect of gp120 coat protein from HIV-1 on tyrosine phosphorylation processes in primary cultures of granular neurons or glial cells from the cerebellum of neonatal rats. The extracellular application of recombinant gp120 (200 pM) was able to reduce the phosphotyrosine content and the immunoreactivity for active form-specific antibodies of MAP kinase. Whereas in neurons MAP kinase appeared to be the only protein whose phosphotyrosine content was decreased, in glial cultures the inhibitory effect of gp120 on tyrosine phosphorylation processes appeared to be more widespread. In neuronal cultures, the effect of the viral protein was prevented by the concomitant treatment with depolarizing agents.

We examined the effect of estrogen on mitogen-activated protein kinase (MAPK) in osteoblastic cells. Rat ROS 17/2.8 cells were exposed to 17beta-estradiol (E2) and MAPK activity in the cells was measured by an in vitro phosphorylation assay. E2 treatment caused a rapid and transient MAPK activation within 5 min. Insulin-like growth factor-I, which acts via their membrane receptors, caused a similar effect, but it required 10 min to reach the maximum level. Western blot analyses with anti-MAPK and anti-phosphotyrosine antibodies demonstrated that the E2 activation of MAPK was accompanied by phosphorylation of the enzyme. The concentration range (10 nM-1 pM) of E2 needed for this MAPK activation was less than that (1 microM-0.1 nM) needed for the transcriptional activation via the nuclear estrogen receptor (ER). These data provide the first evidence of MAPK activation by E2 through phosphorylation, which may be mediated through a putative plasma membrane receptor in the cultured bone cells.

We reported that polyclonal antibody against Ca2+/calmodulin-dependent protein kinase V (CaM kinase V) reacted to two proteins of rat cerebrum with a molecular mass of 40 and 41 kDa. This antibody revealed the immunoreactivity with CaM kinase I expressed in E. coli (recombinant CaM kinase I), of which molecular mass was 40 kDa, whereas 41 kDa mainly with purified CaM kinase V. The immunoreactive bands of recombinant CaM kinase I and CaM kinase V did not shift by phosphorylation or dephosphorylation. These results suggest that CaM kinase V and CaM kinase I may form a family of isoforms.