**5.4. First evidence of rapid nongenomic action of thyroid hormone and its metabolites on the synaptosomal protein phosphorylation in adult rat brain,** *in vitro*

Protein phosphorylation and dephosphorylation are now recognized to be major regulatory mechanisms by which neural activities are controlled by external physiological signals or stimuli. Several nongenomic mechanisms are coordinated by rapid post-transcriptional modifications, such as protein phosphorylation and dephosphorylation reactions, which act like a molecular switch to control intracellular signaling mechanisms. Abnormalities of these imperative regulatory signaling processes produce deleterious effects on the CNS. As a consequence, variety in unusual protein phosphorylation is the end result of many major neuropshychological dysfunctions leading to diseases [78]. Numerous second messenger molecules regulate cellular physiology by effects on protein kinases and phosphatases. Protein kinases catalyze the transfer of the terminal -phosphate group of ATP or GTP to the hydroxyl group of serine, threonine or tyrosine in substrate proteins. Their structure, subcellular localization and substrate specificity allow them to control cellular physiology. These proteins largely make up the cell signaling pathways that transmit, amplify and integrate signals from the extracellular environment. Protein phosphorylation promotes enzyme activation or deactivation. Phosphorylated proteins are substrates for protein phosphatases and dephosphorylation occur to serve as a molecular switch to fine tune a cellular response [79].

Variety of agents regulating the activity of NKA raises the possibility of the NKA as a substrate molecule that is subject to regulation by phosphorylation or dephosphorylation. Indeed, inhibition of NKA is associated with the phosphorylation of the enzyme by both PKA and PKC. This inhibition of NKA has been attributed to the phosphorylation of 1 subunit of the NKA molecule at serine residues by PKA and PKC site-specifically. Isoproterenol (-adrenergic agonist that activates adenylate cyclase to produce cAMP, an activator of PKA), forskolin (adenylate cyclase activator), and okadaic acid (an inhibitor of protein phosphatase-1 and -2A) have been reported to increase significantly the level of phosphorylation of wild-type 1-subunit of the NKA in COS cells, accompanied by a significant inhibition of the enzyme activity [62,63]. Among nine distinct isoforms of adenylate cyclase (AC), three isoforms are Ca2+/calmodulin-dependent, including type I-AC, III-AC [80,81], and VIII-AC. The Ca2+/calmodulin-dependent AC is an integral membrane protein [82]. Hence, one possible role of Ca2+/calmodulin may be to stimulate Ca2+/calmodulin-dependent AC followed by cAMP production and phosphorylation of the NKA, exactly as -adrenergic receptor agonists do.

While a direct effect of TH on protein kinase activity has not been formerly studied in tissues from mature brain, hypothyroidism has been linked with reduced levels of phosphorylated MAPK in the hippocampus [83]. Based on these observations, possibility of a metabotropic pathway for rapid actions of TH on protein phosphorylation in synaptosomes from adult rat brain was investigated.

20 Thyroid Hormone

*vitro*

cellular response [79].

NKA, exactly as -adrenergic receptor agonists do.

our data indirectly support the involvement of second messenger system (cAMP and/or Ca2+) mediated through G protein activation after specific L-T3-membrane receptor interaction. The membrane NKA has been implicated in several aspects of physiologic

Protein phosphorylation and dephosphorylation are now recognized to be major regulatory mechanisms by which neural activities are controlled by external physiological signals or stimuli. Several nongenomic mechanisms are coordinated by rapid post-transcriptional modifications, such as protein phosphorylation and dephosphorylation reactions, which act like a molecular switch to control intracellular signaling mechanisms. Abnormalities of these imperative regulatory signaling processes produce deleterious effects on the CNS. As a consequence, variety in unusual protein phosphorylation is the end result of many major neuropshychological dysfunctions leading to diseases [78]. Numerous second messenger molecules regulate cellular physiology by effects on protein kinases and phosphatases. Protein kinases catalyze the transfer of the terminal -phosphate group of ATP or GTP to the hydroxyl group of serine, threonine or tyrosine in substrate proteins. Their structure, subcellular localization and substrate specificity allow them to control cellular physiology. These proteins largely make up the cell signaling pathways that transmit, amplify and integrate signals from the extracellular environment. Protein phosphorylation promotes enzyme activation or deactivation. Phosphorylated proteins are substrates for protein phosphatases and dephosphorylation occur to serve as a molecular switch to fine tune a

Variety of agents regulating the activity of NKA raises the possibility of the NKA as a substrate molecule that is subject to regulation by phosphorylation or dephosphorylation. Indeed, inhibition of NKA is associated with the phosphorylation of the enzyme by both PKA and PKC. This inhibition of NKA has been attributed to the phosphorylation of 1 subunit of the NKA molecule at serine residues by PKA and PKC site-specifically. Isoproterenol (-adrenergic agonist that activates adenylate cyclase to produce cAMP, an activator of PKA), forskolin (adenylate cyclase activator), and okadaic acid (an inhibitor of protein phosphatase-1 and -2A) have been reported to increase significantly the level of phosphorylation of wild-type 1-subunit of the NKA in COS cells, accompanied by a significant inhibition of the enzyme activity [62,63]. Among nine distinct isoforms of adenylate cyclase (AC), three isoforms are Ca2+/calmodulin-dependent, including type I-AC, III-AC [80,81], and VIII-AC. The Ca2+/calmodulin-dependent AC is an integral membrane protein [82]. Hence, one possible role of Ca2+/calmodulin may be to stimulate Ca2+/calmodulin-dependent AC followed by cAMP production and phosphorylation of the

**5.4. First evidence of rapid nongenomic action of thyroid hormone and its metabolites on the synaptosomal protein phosphorylation in adult rat brain,** *in* 

processes including its role in neurotransmitter release [43].

**Figure 9.** Representative autoradiogram of SDS-PAGE separation of proteins incorporating 32P in the presence of L-T3. Lanes were loaded with synaptosomal lysates which had been preincubated at 0°C for 60 min and 37°C for 5 min with (from left): 1mM Na3VO4 (V), 1, 3, 10, 30, 100, 300, 1000, or 0 (C = control) nM L-T3 and then incubated with 20μM of [γ-32P]-ATP (3 μCi) for 1 min at 37°C. Left panel (a): Silver-stained gel for visualization of protein bands. Right panel (b): Autoradiogram of same gel showing increased incorporation of 32P in four prominent bands (α: 381 kD, β: 531 kD, γ: 631 kD, δ: 1131 kD). (c) Normalized data showing effect of *in vitro* addition of graded doses of L-T3 on the levels of protein phosphorylation expressed as optical density (OD)/protein (Ref. Sarkar et al. 2006 *Neuroscience* 137: 125-132 acknowledged [11]).

Our observation demonstrated that TH induces rapid changes in synaptosomal protein phosphorylation. Incubation with L-T3 or L-T4 specifically showed significant biphasic dose-dependent effects on the phosphorylation of 381, 531, 621, and 1131 kD proteins. *In vitro* brain physiologic concentrations of TH (1-30 nM) showed significant increase in the levels of protein phosphorylation rapidly within minutes (Figure 9). In contrast, incubations with similar doses of reverse-T3 (rT3) were without significant effect, indicating specificity for L-T3 and L-T4. The protein phosphorylation statuses of these four synaptosomal

proteins were significantly increased followed by L-T3 and L-T4 treatment as well. Both L-T3 and L-T4 indicated bi-phasic nature of effect for each of these proteins phosphorylated. Maximum levels of phosphorylation were noticed at concentration range from 10-30 nM. However, no significant effect on protein phosphorylation was observed as an effect of rT3 on any of these proteins. This effect of rT3 clearly confirmed very structural and functional specificity of L-T3 on protein phosphorylation. Determination of time course of protein phosphorylation followed by one single *in vitro* dose of L-T3 showed it peaked rapidly between 180 seconds to 240 seconds and thereafter it decreased. This indicated a rapid action of THs and its metabolites [11].

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 23

TH in mature mammalian brain and provided additional support for the contention that TH

Many nongenomic mechanisms are modulated by phosphorylation–dephosphorylation of substrate proteins. Multiple Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) and Ca2+/phospholipid-dependent protein kinases (PKCs) have been identified in brain. Among these, CaMPK-II is the most abundant Ca2+/CaM-stimulated protein kinase in brain. CaMPK-II is important in several neuronal functions, including neurotransmitter release and the modulation of the functional properties of ion channels and receptors. CaMPK-II is differentially expressed in different brain regions of cells, exists in both cytosolic and membrane-associated forms and is especially concentrated in the postsynaptic density and synaptic vesicles. A distinct property of CaMPK-II is that autophosphorylation of its threonine residue near the calmodulin binding domain converts it to a Ca2+ independent state. Further, it has been shown that calmodulin-dependent autophosphorylation of CaMPK-II induces a conformational changes in the region of the calmodulin binding domain that allows additional stabilizing interactions with calmodulin. This autophosphorylation may involve in extending the effects triggered by a transient calcium signal. PTU-induced mild hypothyroidism in chick brain during posthatch development has been shown to increase the level of Ca2+/CaM-stimulated phosphorylation in cytosol, but lower it in the membrane, indicating a role of thyroid hormones in

The effect of Ca2+ and calmodulin on TH-induced total protein phosphorylation and their regulation was explored. L-T3 significantly and dose-dependently (10 nM-1 M) increased total 32P- incorporation into synaptosomal proteins, *in vitro*, over the basal level of phosphorylation. Although L-T3 exerted its own independent effect on increase in overall total protein phosphorylation, specifically it established its role to be at least dependent on Ca2+ and calmodulin. Ca2+ also showed its independent influence on the basal L-T3-induced total protein phosphorylation in synaptosomal isolates. The dependency of L-T3-induced total synaptosomal protein phosphorylation was evaluated and finally confirmed using EGTA (Ca2+-ion chelator) and KN-62 (a specific blocker of CaMK-II). *In vitro* addition of 10 nM and 100 nM doses of L-T3 alone did not alter significantly the basal levels of phosphorylation. However, the 1 M dose of L-T3 significantly amplified the signal by ~1.3 fold compared to the basal level (P<0.05). Next, we wanted to determine whether Ca2+ augments protein phosphorylation in the presence of L-T3. Ca2+ (0.5 mM) were able to significantly increase the basal phosphorylation level. However, no further significant changes were noticed with additional 10 nM or 100 nM L-T3. However, 1 μM concentration of L-T3 augmented the signal significantly (P<0.05) by ~1.5-fold (0.2167 pmols/min/mg protein) as compared to the Ca2+-treated baseline (0.1475 pmols/min/mg protein), and by ~2.2-fold over the basal phosphorylation (0.097 pmols/min/mg protein). In contrast, the effects of low physiological concentrations of L-T3 were dramatically enhanced when 2 M CaM was added to the Ca2++ L-T3-treatment group. In the presence of Ca2+ and CaM, L-T3

*5.4.1.1. Role of calcium and calmodulin on synaptosomal protein phosphorylation, in vitro* 

has a unique and complex signaling function in adult brain [12].

distributing CaMPK-II during developmental changes [78,86].

5.4.1.1.1. Effect of L-T3 on total protein phosphorylation

Our next interest was to see which amino acids present in these phosphoyraled proteins are targets. Hence phospho-specifc antibodies for tyrosine and serine were used in western bolt analysis. Immunoblot analysis of synaptosomal lysates incubated with L-T3 (1 nM-1 M) confirmed phosphorylation at the seryl residues of a ~112 kD protein and phosphorylation at tyrosyl residues of a distinct ~ 95 kD protein. These data support that THs have a diversity of rapid nongenomic pathways for regulation of protein phosphorylation in mature mammalian brain [11]. Especially, the α-subunit of NKA is a ~112 kD membrane protein. Indeed, inhibition of NKA is associated with the phosphorylation of its subunits by both PKA and PKC. This inhibition of NKA has been attributed to the site-specific phosphorylation of the α1-subunit of the NKA at seryl residues by PKA and PKC [61-63]. In adult rat alveolar epithelial cell L-T3 induced translocation of NKA to plasma membrane. NKA stimulation by L-T3 was assigned to L-T3-induced stimulation of PI3K/PKB pathway via the Src family of tyrosine kinases nongenomically [84]. These data suggest possible involvement of membrane components in TH-induced protein phosphorylation.

Examples of nongenomic control of protein phosphorylation by L-T3 also have been reported in few other tissues. Nongenomic relationship of MAPK and MAPK-mediated protein phosphorylation at the seryl residue of nuclear TH receptor has been described in 293T cells [68]. This indicated a control of nongenomic mechanism on genomic mechanism. In developing brain, inhibition of PKA transcriptionally blocked L-T3-induced actin gene expression, whereas PKC and tyrosine kinase did not influence it significantly [85].
