Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, and its activity is regulated by phosphorylation in the N-terminal regulatory domain. The proline-directed serine/threonine kinase cyclin-dependent kinase 5 (cdk5) plays an important role in diverse neuronal processes. In the present study, we identify TH as a novel substrate of cdk5. We show that cdk5 phosphorylates TH at serine 31 and that this phosphorylation is associated with an increase in total TH activity. In transgenic mice with increased cdk5 activity, the immunoreactivity for phosphorylated TH at Ser-31 is enhanced in neurons of the substantia nigra, a brain region enriched with TH-positive neurons. In addition, we demonstrate that co-expression of cdk5 and its regulatory activator p35 with TH increases the stability of TH. Consistent with these findings, TH protein levels are reduced in cdk5 knock-out mice. Importantly, the TH activity and protein turnover of the phosphorylation-defective mutant TH S31A was not altered by cdk5 activity. Taken together, these data suggest that cdk5 phosphorylation of TH is an important regulator of TH activity through stabilization of TH protein levels.
"Curiously though phosphorylated MAP kinase (pMAPK), the enzyme most commonly responsible for Ser31TH phosphorylation, at least in vitro (Dunkley et al., 2004; Haycock et al., 1992), was not elevated. It is possible that cyclindependent kinase 5 may directly phosphorylate Ser31TH (Moy and Tsai, 2004) in response to HDZ administration. Nevertheless, the blunting of the adrenal medullary response and particularly the lack of activated chromaffin cells following HDZ treatment, which contrasts with the robust activation seen following 2DG administration (Parker et al., 2013; Ritter et al., 1998), may be explained by multiple direct actions that HDZ has on chromaffin cells. "
"It is noteworthy that multiple pieces of evidence suggest an intimate relationship between Cdk5 and the dopamine system. Cdk5 phosphorylates tyrosine hydroxylase (TH), regulating its stability, and thus maintaining dopaminergic homeostasis . In postsynaptic neurons, when the T75 residue of dopamine and cyclic-AMP regulated phosphoprotein-32kD (DARPP-32) is phosphorylated by Cdk5, it can inhibit PKA activity and thus antagonize dopamine DRD1-mediated PKA signaling . "
[Show abstract][Hide abstract] ABSTRACT: The dopamine D2 receptor (DRD2) is a key receptor that mediates dopamine-associated brain functions such as mood, reward, and emotion. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase whose function has been implicated in the brain reward circuit. In this study, we revealed that the serine 321 residue (S321) in the third intracellular loop of DRD2 (D2i3) is a novel regulatory site of Cdk5. Cdk5-dependent phosphorylation of S321 in the D2i3 was observed in in vitro and cell culture systems. We further observed that the phosphorylation of S321 impaired the agonist-stimulated surface expression of DRD2 and decreased G protein coupling to DRD2. Moreover, the downstream cAMP pathway was affected in the heterologous system and in primary neuronal cultures from p35 knockout embryos likely due to the reduced inhibitory activity of DRD2. These results indicate that Cdk5-mediated phosphorylation of S321 inhibits DRD2 function, providing a novel regulatory mechanism for dopamine signaling.
PLoS ONE 12/2013; 8(12):e84482. DOI:10.1371/journal.pone.0084482 · 3.23 Impact Factor
"In keeping with this and our other studies in vivo
,  a change in Ser31 phosphorylation was evident at both 30 and 60 min after the stimulus. Phosphorylation of Ser31 depends upon MAPK , , although CDK5 may also play a direct or indirect (via activation of MEK1) role , , –. In a previous study we have demonstrated that pMAPK is significantly elevated in the rostral C1 and A6 30 min following HDZ  suggesting that the increased pSer31 in these regions may result from activation of MAPK. "
[Show abstract][Hide abstract] ABSTRACT: The expression of c-Fos defines brain regions activated by the stressors hypotension and glucoprivation however, whether this identifies all brain sites involved is unknown. Furthermore, the neurochemicals that delineate these regions, or are utilized in them when responding to these stressors remain undefined. Conscious rats were subjected to hypotension, glucoprivation or vehicle for 30, 60 or 120 min and changes in the phosphorylation of serine residues 19, 31 and 40 in the biosynthetic enzyme, tyrosine hydroxylase (TH), the activity of TH and/or, the expression of c-Fos were determined, in up to ten brain regions simultaneously that contain catecholaminergic cell bodies and/or terminals: A1, A2, caudal C1, rostral C1, A6, A8/9, A10, nucleus accumbens, dorsal striatum and medial prefrontal cortex. Glucoprivation evoked phosphorylation changes in A1, caudal C1, rostral C1 and nucleus accumbens whereas hypotension evoked changes A1, caudal C1, rostral C1, A6, A8/9, A10 and medial prefrontal cortex 30 min post stimulus whereas few changes were evident at 60 min. Although increases in pSer19, indicative of depolarization, were seen in sites where c-Fos was evoked, phosphorylation changes were a sensitive measure of activation in A8/9 and A10 regions that did not express c-Fos and in the prefrontal cortex that contains only catecholaminergic terminals. Specific patterns of serine residue phosphorylation were detected, dependent upon the stimulus and brain region, suggesting activation of distinct signaling cascades. Hypotension evoked a reduction in phosphorylation in A1 suggestive of reduced kinase activity. TH activity was increased, indicating synthesis of TH, in regions where pSer31 alone was increased (prefrontal cortex) or in conjunction with pSer40 (caudal C1). Thus, changes in phosphorylation of serine residues in TH provide a highly sensitive measure of activity, cellular signaling and catecholamine utilization in catecholaminergic brain regions, in the short term, in response to hypotension and glucoprivation.
PLoS ONE 11/2012; 7(11):e50535. DOI:10.1371/journal.pone.0050535 · 3.23 Impact Factor
Rui Zhang, Nan Yang, Chao Ji, Ji Zheng, Zhen Liang, Chun-Ying Hou, Yan-Yong Liu, Ping-Ping Zuo
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