The alpha-synuclein gene is implicated in the pathogenesis of Parkinson's disease. Although alpha-synuclein function is uncertain, the protein has homology to the chaperone molecule 14-3-3. In addition, alpha-synuclein can bind to 14-3-3, and both alpha-synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) biosynthesis, we explored whether alpha-synuclein also bound to tyrosine hydroxylase and influenced its activity. Immunoprecipitation revealed an interaction between alpha-synuclein and tyrosine hydroxylase in brain homogenates and MN9D dopaminergic cells. Colocalization of alpha-synuclein with tyrosine hydroxylase was confirmed by immunoelectron microscopy. To explore the consequences of the interaction, we measured the effect of recombinant alpha-synuclein on tyrosine hydroxylase activity in a cell-free system and observed a dose-dependent inhibition of tyrosine hydroxylase by alpha-synuclein. To measure the impact of alpha-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably transfected MN9D cells with wild-type or A53T mutant alpha-synuclein. Overexpression of wild-type or A53T mutant alpha-synuclein did not significantly alter tyrosine hydroxylase protein levels in our stably transfected cells. However, overexpressing cell lines had significantly reduced tyrosine hydroxylase activity and a corresponding reduction in dopamine synthesis. The reduction in cellular dopamine levels was not caused by increased dopamine catabolism or dopamine efflux. These data suggest that alpha-synuclein plays a role in the regulation of dopamine biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If so, a loss of soluble alpha-synuclein, by reduced expression or aggregation, could increase dopamine synthesis with an accompanying increase in reactive dopamine metabolites.

Altered neural-endocrine relations have been proposed as factors in mammalian aging. In the same rats from three age groups we quantified astrocyte reactivity in hippocampus, performed radioimmunoassays for plasma adrenocorticoids, and measured adrenal weight. These variables were correlated in individual animals and generally increased with age. The findings are consistent with recent hypotheses that endocrine levels are related to brain aging, either as cause or effect.

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca 21 -dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative in¯uence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K 1 -stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-de®cient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using siteand phosphorylation state-speci®c antibodies. Treatment with elevated K 1 (158 mM) for 5 min increased TH PS at each site in a Ca 21 -dependent manner. Pretreatment with PD98059 prevented elevated K 1 -stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not signi®cantly increased by forskolin, and elevated K 1 -stimulated Ser40 PS was three-to ®ve-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K 1 and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS. Keywords: blot immunolabeling, Ca 21 /calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, extracellular signal-regulated protein kinase, PC12, PD98059.

We have identified the beta (beta) isoform of the 14‐3‐3 family of proteins as an activator of the Raf‐1 protein kinase. 14‐3‐3 was isolated in a yeast two‐hybrid screen for Raf‐1 kinase domain binding proteins. Purified bovine brain 14‐3‐3 interacted specifically with both c‐Raf‐1 and the isolated Raf‐1 kinase domain. Association was sensitive to the activation status of Raf‐1; 14‐3‐3 bound to unactivated Raf‐1, but not Raf‐1 activated by protein kinase C alpha or Ras and Lck. The significance of these interactions under physiological conditions was demonstrated by co‐immunoprecipitation of Raf‐1 and 14‐3‐3 from extracts of quiescent, but not mitogen‐stimulated, NIH 3T3 cells. 14‐3‐3 was not a preferred Raf‐1 substrate in vitro and did not significantly affect Raf‐1 kinase activity in a purified system. However, in cell‐free extracts 14‐3‐3 acted as a Ras‐independent activator of both c‐Raf‐1 and the Raf‐1 kinase domain. The same results were obtained in vivo using transfection assays; 14‐3‐3 enhanced both c‐Raf‐1‐ and Raf‐1 kinase domain‐stimulated expression of AP‐1‐ and NF‐kappa B‐dependent reporter genes and accelerated Raf‐1 kinase domain‐triggered differentiation of PC12 cells. We conclude that 14‐3‐3 is a latent co‐activator bound to unactivated Raf‐1 in quiescent cells and mediates mitogen‐triggered but Ras‐independent regulatory effects aimed directly at the kinase domain.

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