Living cells are adaptive self-sustaining systems. They strictly depend on the sufficient supply of oxygen, energy, and nutrients from the outside in order to sustain their internal organization. However, as autonomous entities they are able to monitor and appropriately adapt to any critical fluctuation in their environment. In the case of insufficient external nutrient supply or augmented energy demands, cells start to extensively digest their own interior. This process, known as macroautophagy, comprises the transport of cytosolic portions and entire organelles to the lysosomal compartment via specific double-membrane vesicles, called autophagosomes. Although extensively upregulated under nutrient restriction, a low level of basal autophagy is likewise crucial in order to sustain the cellular homeostasis. On the other hand, cells have to avoid excessive and enduring self-digestion. The delicate balance between external energy and nutrient supply and internal production and consumption is a demanding task. The complex protein network that senses and precisely reacts to environmental changes is thus mainly regulated by rapid and reversible posttranslational modifications such as phosphorylation. This review focuses on the serine/threonine protein kinases AMP-activated protein kinase, mammalian target of rapamycin (mTOR), and unc-51-like kinase 1/2 (Ulk1/2), three interconnected major junctions within the autophagy regulating signaling network. AMPK: THE ENERGY-SENSING KINASEA MP-activated protein kinase (AMPK) was initially identified as a serine/threonine kinase that negatively regulates several key enzymes of the lipid anabolism (30). Meanwhile, AMPK is regarded as the major energy-sensing kinase that activates a whole variety of catabolic processes in multicellular organisms such as glucose uptake and metabolism, while simultaneously inhibiting several anabolic pathways, such as lipid, protein, and carbohydrate biosynthesis (reviewed in reference 30).AMPK is a heterotrimeric protein complex that is precisely regulated in different ways. First, the phosphorylation of a conserved threonine residue (T172) in the activation loop of the catalytic ␣-subunit by upstream kinases is a prerequisite for the activity of AMPK. Several AMPK-phosphorylating kinases have been identified thus far. In addition to the ubiquitously expressed and constitutively active kinase LKB1 (31, 109), Ca 2ϩ -activated Ca 2ϩ /calmodulin-dependent kinase kinase ␤ (CaMKK␤) (32,43,108) and transforming growth factor ␤-activated kinase-1 (TAK1) (80) are both known as activators of AMPK. Second, AMPK activity can further be modulated by allosteric binding to the regulatory ␤-and ␥-subunit. Since the ratio of AMP to ATP represents the most accurate way to precisely measure the intracellular energy level, both AMP and ATP are able to oppositely regulate the activity of AMPK. While AMP binding to the ␥-subunit allosterically enhances AMPK kinase activity and prevents the dephosphorylation of T172, ATP is known to counteract the activating properties...

Unc-51-like kinase 1 (Ulk1) plays a central role in autophagy induction. It forms a stable complex with Atg13 and focal adhesion kinase (FAK) family interacting protein of 200 kDa (FIP 200). This complex is negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1) in a nutrient-dependent way. AMP-activated protein kinase (AMPK), which is activated by LKB1/Strad/Mo25 upon high AMP levels, stimulates autophagy by inhibiting mTORC1. Recently, it has been described that AMPK and Ulk1 interact and that the latter is phosphorylated by AMPK. This phosphorylation leads to the direct activation of Ulk1 by AMPK bypassing mTOR-inhibition. Here we report that Ulk1/2 in turn phosphorylates all three subunits of AMPK and thereby negatively regulates its activity. Thus, we propose that Ulk1 is not only involved in the induction of autophagy, but also in terminating signaling events that trigger autophagy. In our model, phosphorylation of AMPK by Ulk1 represents a negative feedback circuit.

Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok-3 (downstream of kinase-3) as a key component of negative feedback regulation in Ca2+ signaling from the B-cell antigen receptor. Dok-3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok-3 inhibits antigen receptor-induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Bruton's tyrosine kinase. This leads to diminished activation of phospholipase C-gamma2 and reduced production of soluble inositol trisphosphate. Hence, the Dok-3/Grb2 module is a membrane-associated signaling organizer, which orchestrates the interaction efficiency of Ca2+-mobilizing enzymes.

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Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase familyinteracting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.