Biomedical subjects

Larisa Bobrovskaya

Publications and source records attributed to Larisa Bobrovskaya.

7 recordsLinked to original sources

PubMed2006

Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesis.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine synthesis. Its activity is known to be controlled acutely (minutes) by phosphorylation and chronically (days) by protein synthesis. Using bovine adrenal chromaffin cells we found that nicotine, acting via nicotinic receptors, sustained the phosphorylation of TH at Ser40 for up to 48 h. Nicotine also induced sustained activation of TH, which for the first 24 h was completely independent of TH protein synthesis, and the phosphorylation of TH at Ser31. Imipramine did not inhibit the acute phosphorylation of TH at Ser40 or TH activation induced by nicotine, but did inhibit the sustained responses to nicotine seen at 24 h. The protein kinase(s) responsible for TH phosphorylation at Ser40 switched from being protein kinase C (PKC) independent in the acute phase to PKC dependent in the sustained phase. Sustained phosphorylation and activation of TH were also observed with histamine and angiotensin II. Sustained phosphorylation of TH at Ser40 provides a novel mechanism for increasing TH activity and this leads to increased catecholamine synthesis. Sustained phosphorylation of TH may be a selective target for drugs or pathology in neurons that contain TH and synthesize dopamine, noradrenaline or adrenaline.

Adrenal Glands↗

PubMed2006

Differential regulation of the human tyrosine hydroxylase isoforms via hierarchical phosphorylation.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline, and adrenaline. In response to short term stimuli TH activity is primarily controlled by phosphorylation of serine 40. We have previously shown that phosphorylation of serine 19 in TH can indirectly activate TH via a hierarchical mechanism by increasing the rate of phosphorylation of serine 40. Here we show that phosphorylation of serine 31 in rat TH increases the rate of serine 40 phosphorylation 9-fold in vitro. Phosphorylation of serine 31 in intact bovine chromaffin cells potentiated the forskolin-induced increase in serine 40 phosphorylation and TH activity more than 2-fold. Humans are unique in that they contain four TH isoforms but to date no significant differences have been shown in the regulation of these isoforms. Phosphorylation of the human TH isoform 1 at serine 31 by extracellular signal-regulated protein kinase (ERK) also produced a 9-fold increase in the rate of phosphorylation of serine 40, whereas little effect was seen in the TH isoforms 3 and 4. ERK did not phosphorylate human TH isoform 2. The effect of serine 19 phosphorylation on serine 40 (44 in TH2) phosphorylation is stronger in TH2 than in TH1. Thus hierarchical phosphorylation provides a mechanism whereby the two major human TH isoforms (1 and 2) can be differentially regulated with only isoform 1 responding to the ERK pathway, whereas isoform 2 is more sensitive to calcium-mediated events.

Amino Acid Sequence↗

PubMed2004

S100B protein stimulates calcineurin activity.

S100B is a calcium binding protein from astrocytes that regulates protein phosphorylation by binding to substrates and protein kinases. S100B might also regulate protein phosphatases and this was investigated for protein phosphatase 2B (calcineurin). The results indicate that S100B (5-10 microM) increased the activity of both purified and cytoskeletal calcineurin in a Ca-dependent manner. This effect was blocked by a specific inhibitor of calcineurin activity, but not by TRTK-12 (an inhibitor of S100B binding to other protein targets). The present results and the known co-localization of S100B and calcineurin in the astrocyte cytoskeleton suggest that S100B may play a role in the phosphorylation state of cytoskeletal proteins.

Animals↗

PubMed2004

Angiotensin II regulates tyrosine hydroxylase activity and mRNA expression in rat mediobasal hypothalamic cultures: the role of specific protein kinases.

Dopamine secreted by hypothalamic neurons is crucial in regulating prolactin secretion from the pituitary. We have examined the ability of angiotensin II (AngII) to regulate the activity of these dopaminergic neurons and thus act as a potential physiological regulator of prolactin secretion. Using a hypothalamic cell culture preparation we determined the effect of AngII on tyrosine hydroxylase activity and expression (TOH). This is important because TOH is the rate-limiting enzyme in dopamine biosynthesis. AngII stimulated a time- and concentration-dependent increase in TOH activity which was suppressed by inhibitors able to act on protein kinase A (PKA), protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (CaMPKII). An inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD 98059, reduced basal TOH activity but the AngII response was still detectable. AngII stimulation enhanced the phosphorylation of TOH at Ser19, Ser31 and Ser40. AngII also induced a time-dependent increase in TOH mRNA expression which was unaffected by inhibitors able to act on PKA and CaMPKII, but was abolished by inhibitors able to act on ERK and PKC. AngII responses were very much larger in cultures prepared from female when compared to male rat pups. Data from adult hypothalamic slices confirmed this sexual dimorphism and supported the role of the protein kinases noted above. Therefore AngII can regulate both the activity and expression of TOH in hypothalamic neurons employing multiple, but only partially overlapping, signaling pathways.

Angiotensin II↗

PubMed2004

Phosphorylation of Ser19 increases both Ser40 phosphorylation and enzyme activity of tyrosine hydroxylase in intact cells.

We have previously shown that the phosphorylation of Ser19 in tyrosine hydroxylase can increase the rate of phosphorylation of Ser40 in tyrosine hydroxylase threefold in vitro. In this report we investigated the role of Ser19 on Ser40 phosphorylation in intact cells. Treatment of bovine chromaffin cells with anisomycin produced a twofold increase in Ser19 phosphorylation with no increase in Ser31 phosphorylation and only a small increase in Ser40 phosphorylation. Treatment of bovine chromaffin cells with forskolin produced a fourfold increase in Ser40 phosphorylation but no significant increase in either Ser19 or Ser31 phosphorylation. When chromaffin cells were first treated with anisomycin, the level of Ser40 phosphorylation after treatment by forskolin was 76% greater than the level of Ser40 phosphorylation in cells treated with forskolin alone. This potentiation of Ser40 phosphorylation by anisomycin could be completely blocked by the p38 MAP (mitogen-activated protein) kinase inhibitor SB 203580. The potentiation of Ser40 phosphorylation by anisomycin was not due to an increase in Ser40 kinase activity. Anisomycin treatment of chromaffin cells potentiated the forskolin-induced increase in tyrosine hydroxylase activity by 50%. This potentiation of activity was also blocked by SB 203580. These data provide the first evidence that the phosphorylation of Ser19 can potentiate the phosphorylation of Ser40 and subsequent activation of tyrosine hydroxylase in intact cells.

Animals↗

PubMed2004

Tyrosine hydroxylase phosphorylation: regulation and consequences.

The rate-limiting enzyme in catecholamine synthesis is tyrosine hydroxylase. It is phosphorylated at serine (Ser) residues Ser8, Ser19, Ser31 and Ser40 in vitro, in situ and in vivo. A range of protein kinases and protein phosphatases are able to phosphorylate or dephosphorylate these sites in vitro. Some of these enzymes are able to regulate tyrosine hydroxylase phosphorylation in situ and in vivo but the identity of the kinases and phosphatases is incomplete, especially for physiologically relevant stimuli. The stoichiometry of tyrosine hydroxylase phosphorylation in situ and in vivo is low. The phosphorylation of tyrosine hydroxylase at Ser40 increases the enzyme's activity in vitro, in situ and in vivo. Phosphorylation at Ser31 also increases the activity but to a much lesser extent than for Ser40 phosphorylation. The phosphorylation of tyrosine hydroxylase at Ser19 or Ser8 has no direct effect on tyrosine hydroxylase activity. Hierarchical phosphorylation of tyrosine hydroxylase occurs both in vitro and in situ, whereby the phosphorylation at Ser19 increases the rate of Ser40 phosphorylation leading to an increase in enzyme activity. Hierarchical phosphorylation depends on the state of the substrate providing a novel form of control of tyrosine hydroxylase activation.

Animals↗

PubMed2002

Lead-stimulated p38MAPK-dependent Hsp27 phosphorylation.

Lead (Pb2+) is a cytotoxic metal ion whose mechanism of action is not established. However, Pb2+ is known to interact with a wide variety of molecules involved in signal transduction. In this study the effect of Pb2+ on protein phosphorylation in bovine adrenal chromaffin cells and human SH SY5Y cells was examined. Cells were incubated with 32P(i) for 1 h in the presence of Pb2+ (1-10 microM) and the proteins were separated by two-dimensional PAGE. An increase in the phosphorylation of a number of proteins was observed in response to Pb2+, including three spots, MW 25 kDa, and pI's in the range 4.0-4.5. These proteins were immunoidentified as three isoforms of the heat-shock protein 27 kDa (Hsp27), and the identity of the most basic spot was confirmed by amino acid sequencing. Phosphorylation of p38MAPK was increased by Pb2+ and the effect of Pb2+ on Hsp27 phosphorylation was blocked by the p38MAPK inhibitor SB203580 (1 microM). The results were similar for bovine chromaffin cells and human SH SY5Y cells. This is the first report showing that Pb2+ can modulate the phosphorylation state of Hsp27 via activation of the p38MAPK pathway.

Animals↗