Phosphorylation of HSL

Phosphorylation of partially purified HSL by PKA, leading to a moderate activation of its activity in vitro, was described in the early 1970s [228, 229] and later confirmed [225, 230, 231]. Subsequent phosphopeptide mapping and phosphoami-no acid analysis suggested that HSL was phosphorylated on a single serine residue, named the regulatory site [232]. Partial amino acid sequencing of phospho-peptides generated from bovine HSL [233], together with determination of the primary structure of rat HSL [234], allowed identification of Ser-563 as the regulatory site [235]. However, more recent data have dramatically challenged this view on the short-term regulation of HSL. The finding that mutation of Ser-563 did not abolish PKA-induced activation of HSL led to the identification of two novel PKA sites, Ser-659 and Ser-660, that seem to be responsible in intact primary adipocytes for the activation of HSL in response to isoproterenol stimulation [236]. The role of phosphorylation of Ser-563 remains elusive (Fig. 11.5).

Apart from three PKA phosphorylation sites described above, HSL is phosphory-lated in vivo in hormonally quiescent cells at a site named the basal site [235], corresponding to Ser-565 in rat HSL [232, 234], i.e. two residues carboxy-terminal to Ser-563. Glycogen synthase kinase-4 [237], Ca2+/calmodulin-dependent kinase II, and AMP-activated protein kinase (AMPK) [238] phosphorylate Ser-565 in vitro without any direct effect on enzyme activity (Fig. 11.5). AMPK has been proposed to be the physiologically relevant kinase [238], based on its involvement in other aspects of lipid metabolism and on its proposed role as fuel gauge [239]. Furthermore, because phosphorylation by AMPK prevented subsequent phosphorylation of Ser-563, and vice versa, it was proposed that phosphorylation of Ser-565 exerts an antili-polytic role [238]. This proposal is supported by experiments showing that preincu-

Adipose Tissue Lipolysis Hsl Ampk

Fig. 11.5 Working model for the interplay be- phatases involved. See text for details. tween basal and regulatory phosphorylation Adapted from Ref. [557] with modifications.

sites at HSL, including the kinases and phos-

Fig. 11.5 Working model for the interplay be- phatases involved. See text for details. tween basal and regulatory phosphorylation Adapted from Ref. [557] with modifications.

sites at HSL, including the kinases and phos-

bation with AICAR (5-aminoimidazole-4-carboxamide 1-/5-D-ribofuranoside) - an activator of AMPK - causes a moderate reduction of the lipolytic response to catecholamines in primary adipocytes [240, 241]. The data indicating mutually exclusive phosphorylation at the basal and regulatory sites imply that activation of HSL by PKA has to be preceded by dephosphorylation of the basal site. Whether this dephos-phorylation is an important regulatory step or merely a result of a high constitutive protein phosphatase activity in the cell is not clear. Moreover, the recent finding that Ser-563 is not essential for HSL activation [236] raises some questions regarding this antilipolytic role of Ser-565 phosphorylation. Also, the moderate antilipolytic effect of AICAR should be interpreted with caution because this substance has many cellular effects and has been questioned as a specific AMPK activator [242].

In fact, recent studies have elucidated completely novel aspects of AMPK physiology as it may be directly involved in the ^-adrenergic regulation of lipolysis. Much experimental evidence has promoted the idea that AMPK acts as an intracellular energy sensor, stimulated by the increased intracellular AMP/ATP ratio when cells are stressed by conditions such as hypoxia/ischemia in the heart and excessive contraction in skeletal muscle. Activated AMPK accelerates ATP-produc-ing pathways, such as fatty acid and glucose oxidation, while reducing ATP consumption, ultimately leading to the preservation or restoration of adequate high energy phosphates [243]. Whereas the importance of AMPK to control lipid meta-

bolism in liver and muscle is well established, its role in regulating lipolysis in adipose tissue has remained controversial. Previous observations led to the hypothesis that AMPK antagonizes lipolysis in adipocytes (see above), presumably to prevent futile cycling and depletion of ATP during unrestrained simultaneous lipolysis and re-esterification. However, subsequent mutational analysis of the PKA phosphorylation sites of HSL raised serious doubts on the negative effect of AMPK phosphorylation on PKA-induced HSL activation [236], which were then strengthened by the finding that isoproterenol stimulated AMPK phosphorylation and activity in isolated rat adipocytes, in apparent contradiction to an anti-lipolytic role for AMPK [244]. Confirming and extending the latter study, it was demonstrated recently, that treatment of murine cultured 3T3-L1 adipocytes with isopro-terenol or forskolin promoted the phosphorylation of AMPK at a critical activating Thr172 residue in a concentration- and time-dependent fashion [245]. This correlated well with stimulation of AMPK activity as measured in the immune complex. Analogs of cAMP mimicked the effect of isoproterenol and forskolin on AMPK phosphorylation. Treatment of the adipocytes with insulin reduced both basal and forskolin-induced AMPK phosphorylation via a pathway dependent on PI-3'K. Overexpression of a dominant-inhibitory mutant of AMPK blocked isoprotere-nol-induced lipolysis by about 50% [245]. These data indicate that in adipocytes a novel pathway operates through which cAMP can lead to the activation of AMPK, and that this pathway is required for maximal stimulation of lipolysis. However, overexpression of a constitutively active AMPK version had a minimal effect on both basal and isoproterenol-induced lipolysis only, which may indicate that AMPK is required but not sufficient for lipolysis induction (in the absence of elevated cAMP). Moreover, it remains unclear from this study how agents that increase cAMP or the nucleotide itself activate AMPK in intact cells. Thr172, the phosphorylation site on AMPK responsible for most of the increase in AMPK activity, is not a consensus PKA phosphorylation site, suggesting that PKA regulates AMPK activity through an indirect mechanism, such as phosphorylating and activating an unknown upstream AMPK kinase. Alternatively, the effect of cAMP could be independent of PKA. The other open question is how AMPK regulates lipolysis. As already mentioned, phosphorylation of HSL at Ser563, Ser659, and Ser 660 by PKA alone does not account for the maximal activity of HSL as in vitro PKA phosphorylation causes a 1.5- to 2.2-fold increase in HSL lipase activity only while isoproterenol stimulates FA release from adipocytes by more than 50-fold. AMPK phosphorylates Ser565, which is located in the regulatory domain of HSL, as are the PKA phosphorylation sites, in vitro. Phosphorylation of Ser565 by AMPK might, speculatively, be involved in regulation of the translocation of HSL to the LD (see below). This would explain the recent finding that mutation of this residue abrogated the ability of HSL to translocate to LD [246]. In addition, or alternatively, several other proteins, participation of which in lipolysis has been documented, such as perilipin and lipotransin (see below), may represent direct or indirect targets of AMPK. Taken together, the available experimental evidence hints at a positive modulatory role of AMPK in /-adrenergic stimulation of lipoly-sis, during which it undergoes phosphorylation and activation and then possibly phosphorylates HSL at Ser565, leading to its translocation to LD. Physiologically, this putative mechanism nicely fits the commonly accepted role of AMPK as intracellular energy sensor since it guarantees that ATP depletion in adipocytes and in other tissues coordinated via the ^-adrenergic response leads to enhanced lipolysis, resulting in the release of FA that can be oxidized by target tissues to compensate for the increased demand for high energy phosphates.

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