MIN6 is one of the few pancreatic ␤ cell lines that respond to physiological concentrations of glucose by secreting insulin, and little is known about the triggered molecular mechanisms. We report below that the response to glucose in the MIN6 cells includes an activation of the p42 and p44 mitogen-activated protein (MAP) kinases (ERK2 and ERK1). This activation also occurred with the antidiabetic sulfonylurea glibenclamide and kainate, a specific agonist of a subtype of the ionotropic glutamate receptors, which depolarize the cytoplasmic membrane. The requirement for a calcium entry through the L-type voltage-gated channels and other characteristics of the regulation of the MAP kinase activity, such as the effect of the elevation of the cAMP concentration by forskolin, were similar to those of the secretion of insulin. However, the activation of the MAP kinases is not required for the secretion of insulin, inasmuch as this effect of glucose was not abolished when the MAP kinases were prevented from activation by PD098059, an inhibitor of the MAP kinase kinase. However, as the MAP kinases were translocated into the nucleus, they might be implicated in the calcium-dependent transcriptional response of the cells to glucose and thus regulate the expression of the insulin gene.

Detection of apoptotic neurons and microglial cells in the brains of human immunodeficiency virus type 1 (HIV-1)-infected patients has suggested that programmed cell death may be implicated in the physiopathology of HIV-1 encephalopathy. To analyze in vitro the intracellular signals induced by HIV-1 in human neurons and the associated neuronal death, we tested cultured human central nervous system (CNS) cells for apoptosis induced by HIV-1 and gp120 and for signaling pathways activated by gp120. HIV-1 and gp120 induced apoptosis of neurons and microglial cells but not of astrocytes or transformed microglial cells. Gp120 activated c-Jun N-terminal kinase (JNK) and p42 extracellular-regulated kinase (ERK) in primary CNS cells, with an early peak of activation at 2 to 5 minutes that was not present when pure microglial or astrocyte cultures were tested, followed by a late and sustained activation (10 and 60 minutes) in primary and enriched glial cell cultures as well as in transformed microglial cells. This demonstrates that gp120 could be an effector of HIV-1-induced apoptosis in the CNS and act directly on neuronal and glial cells.

Mitogen‐activated protein (MAP) kinases have been implicated in multiple responses to extracellular stimuli. In this study we show that MAP kinase activity is enhanced after a KCI pulse. This activation correlates with an increased tyrosine phosphorylation of a 42‐kDa protein as determined by antiphosphotyrosine immunoblot. The same band is found in an anti‐MAP kinase immunoblot. Activity is enhanced within 1 min, reaches a maximum at 2 min, and returns to basal level after 10 min. A second peak of activity is observed between 12 and 30 min. The activation is completely blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), showing the involvement of the AMPA type of glutamate receptor. Partial inhibition of MAP kinase activation by 2‐amino‐5‐phosphonovalerate (APV) also shows the involvement of the NMDA receptor. Because the KCI pulse used induces long‐term potentiation (LTP) in rat hippocampal slice, we conclude that MAP kinase may be involved in neuronal transduction events leading to LTP.

The src family protein tyrosine kinases (PTKs) are nonreceptor kinases. Some PTKs of this family are ubiquitously expressed, whereas others have a more restricted expression, as in neurons. Lymphoid cell kinase (lck) p56lck is highly expressed in tissues of lymphoid origin and believed to be specific for hematopoietic cells. Reports suggesting that CD4 is expressed in neurons prompted us to analyze the possibility that p56lck is also expressed in these cells. By western blot and immunoprecipitations using anti‐lck antibody, an lck‐like protein was detected in lysates from primary cultures of rat cerebellar granular neurons. This 56‐kDa phosphoprotein was autophosphorylated in vitro and also phosphorylated enolase, similarly to p56lck. It was shown to be located actually in the neurons by immunocytofluorescence. Partial proteolysis mapping showed that the 56‐kDa phosphoprotein had a peptide pattern very similar to the p56lck protein. Retrotranscription‐PCR allowed the detection of an lck RNA in the neurons. The lck kinase domain was completely identical to the lymphocyte lck kinase domain, but the 5′ end was modified in the neurons. These results show that p56lck is not lymphoid specific as is widely believed; its expression in neurons might underlie the toxicity of the HIV glycoprotein gp120 to neurons.

p56lck, a p60src related tyrosine protein kinase was recently described in a murine lymphoma cell line (LSTRA), in several human lymphomas and in normal peripheral lymphocytes. We have purified p56lck to homogeneity by electrofocusing followed by two-step SDS/polyacrylamide gel electrophoresis. The pure protein was identical to the p56 characterized in LSTRA cells and used to raise antibodies in rabbits. This simple procedure is applicable for the rapid purification of minor proteins.

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