Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser⁴¹ of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca²⁺ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser⁴¹. This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca²⁺ homeostasis.     

Phosphorylation of Zona Occludens-2 by Protein Kinase Cε Regulates Its Nuclear Exportation

Here, we have analyzed the subcellular destiny of newly synthesized tight junction protein zona occludens (ZO)-2. After transfection in sparse cells, 74% of cells exhibit ZO-2 at the nucleus, and after 18 h the value decreases to 17%. The mutation S369A located within the nuclear exportation signal 1 of ZO-2 impairs the nuclear export of the protein. Because Ser369 represents a putative protein kinase C (PKC) phosphorylation site, we tested the effect of PKC inhibition and stimulation on the nuclear export of ZO-2. Our results strongly suggest that the departure of ZO-2 from the nucleus is regulated by phosphorylation at Ser369 by novel PKCε. To test the route taken by ZO-2 from synthesis to the plasma membrane, we devised a novel nuclear microinjection assay in which the nucleus served as a reservoir for anti-ZO-2 antibody. Through this assay, we demonstrate that a significant amount of newly synthesized ZO-2 goes into the nucleus and is later relocated to the plasma membrane. These results constitute novel information for understanding the mechanisms that regulate the intracellular fate of ZO-2.     

Apoptotic Phosphorylation of Histone H3 on Ser-10 by Protein Kinase C��

Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC) δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new ‘apoptotic histone code’ mediated by PKCδ.     

Phosphorylation of ��6-Tubulin by Protein Kinase C�� Activates Motility of Human Breast Cells

Engineered overexpression of protein kinase Cα (PKCα) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCα (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364–2370) revealed that α6-tubulin is phosphorylated in cells expressing traceable PKCα and in vitro by wild type PKCα. Gain-of-function, single site mutations (Ser → Asp) were constructed at each PKC consensus site in α6-tubulin (Ser¹⁵⁸, Ser¹⁶⁵, Ser²⁴¹, and Thr³³⁷) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser¹⁶⁵ as the only site in α6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCα. Expression of a phosphorylation-resistant mutant (S165N-α6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCα or by treatment with PKCα-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous α-tubulin that could be blocked when S165N-α6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCα levels (MDA-MB-231 cells) or lack PKCα and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type α6-tubulin and S165N-α6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser¹⁶⁵ is also a major site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazole-sensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates α-tubulin at Ser¹⁶⁵, leading to microtubule elongation and motility.     

The Nuclear Localization of γ-Tubulin Is Regulated by SadB-mediated Phosphorylation

γ-Tubulin is an important cell division regulator that arranges microtubule assembly and mitotic spindle formation. Cytosolic γ-tubulin nucleates α- and β-tubulin in a growing microtubule by forming the ring-shaped protein complex γTuRC. Nuclear γ-tubulin also regulates S-phase progression by moderating the activities of E2 promoter-binding factors. The mechanism that regulates localization of γ-tubulin is currently unknown. Here, we demonstrate that the human Ser/Thr kinase SadB short localizes to chromatin and centrosomes. We found that SadB-mediated phosphorylation of γ-tubulin on Ser³⁸⁵ formed chromatin-associated γ-tubulin complexes that moderate gene expression. In this way, the C-terminal region of γ-tubulin regulates S-phase progression. In addition, chromatin levels of γ-tubulin were decreased by the reduction of SadB levels or expression of a non-phosphorylatable Ala³⁸⁵-γ-tubulin but were enhanced by expression of SadB, wild-type γ-tubulin, or a phosphomimetic Asp³⁸⁵-γ-tubulin mutant. Our results demonstrate that SadB kinases regulate the cellular localization of γ-tubulin and thereby control S-phase progression.     

Rapid Production of Platelet-activating Factor Is Induced by Protein Kinase Cα-mediated Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 Protein

Platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is synthesized rapidly in response to extracellular stimuli by the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). We have reported previously that lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: rapid activation (30 s) after PAF stimulation and minutes to hours after LPS stimulation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) was later identified as a Ca²⁺-dependent lyso-PAFAT. However, the mechanism of rapid lyso-PAFAT activation within 30 s has not been elucidated. Here we show a new signaling pathway for rapid biosynthesis of PAF that is mediated by phosphorylation of LPCAT2 at Ser-34. Stimulation by either PAF or ATP resulted in PKCα-mediated phosphorylation of LPCAT2 to enhance lyso-PAFAT activity and rapid PAF production. Biochemical analyses showed that the phosphorylation of Ser-34 resulted in augmentation of V_max with minimal K_m change. Our results offer an answer for the previously unknown mechanism of rapid PAF production.     

Phosphorylation of Rab5a Protein by Protein Kinase C? Is Crucial for T-cell Migration

Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase Cϵ (PKCϵ) in migrating T-cells. After stimulation of T-cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on an N-terminal Thr-7 site by PKCϵ. Both Rab5a and PKCϵ dynamically interact at the centrosomal region of migrating cells, and PKCϵ-mediated phosphorylation on Thr-7 regulates Rab5a trafficking to the cell leading edge. Furthermore, we demonstrate that Rab5a Thr-7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement, and T-cell motility. We present a novel mechanism by which a PKCϵ-Rab5a-Rac1 axis regulates cytoskeleton remodeling and T-cell migration, both of which are central for the adaptive immune response.     

Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates ��2 Adrenoceptor Coupling to Gi Proteins in Cardiomyocytes

Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. β₂ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G_s and G_i proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that the β₂AR coupling to G_i proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the β₂AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G_i inhibitor pertussis toxin (PTX). Further studies revealed that activated β₂ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the β₂AR blocked receptor/G_i coupling. However, a sufficient β₂AR/G_i coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (≥100 nm). Disruption of the GRK phosphorylation sites on the β₂AR blocked receptor internalization and coupling to G_i proteins, probably by preventing the receptor''s transportation to access G_i proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G_i-specific inhibitor, G_iCT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the β₂AR for agonist dose-dependent coupling to G_i proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.     

Grb2 Promotes Integrin-Induced Focal Adhesion Kinase (FAK) Autophosphorylation and Directs the Phosphorylation of Protein Tyrosine Phosphatase α by the Src-FAK Kinase Complex

The integrin-activated Src-focal adhesion kinase (FAK) kinase complex phosphorylates PTPα at Tyr789, initiating PTPα-mediated signaling that promotes cell migration. Recruitment of the BCAR3-Cas complex by PTPα-phospho-Tyr789 at focal adhesions is one mechanism of PTPα signaling. The adaptor protein Grb2 is also recruited by PTPα-phospho-Tyr789, although the role of the PTPα-Grb2 complex in integrin signaling is unknown. We show that silencing Grb2 expression in fibroblasts abolishes PTPα-Tyr789 phosphorylation and that this is due to two unexpected actions of Grb2. First, Grb2 promotes integrin-induced autophosphorylation of FAK-Tyr397. This is impaired in Grb2-depleted cells and prohibits FAK activation and formation of the Src-FAK complex. Grb2-depleted cells contain less paxillin, and paxillin overexpression rescues FAK-Tyr397 phosphorylation, suggesting that the FAK-activating action of Grb2 involves paxillin. A second distinct role for Grb2 in PTPα-Tyr789 phosphorylation involves Grb2-mediated coupling of Src-FAK and PTPα. This requires two phosphosites, FAK-Tyr925 and PTPα-Tyr789, for Grb2-Src homology 2 (SH2) binding. We propose that a Grb2 dimer links FAK and PTPα, and this positions active Src-FAK in proximity with other, perhaps integrin-clustered, molecules of PTPα to enable maximal PTPα-Tyr789 phosphorylation. These findings identify Grb2 as a new FAK activator and reveal its essential role in coordinating PTPα tyrosine phosphorylation to enable downstream integrin signaling and migration.     

A Biologically Active Sequence of the Laminin ��2 Large Globular 1 Domain Promotes Cell Adhesion through Syndecan-1 by Inducing Phosphorylation and Membrane Localization of Protein Kinase C��

Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 α2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin α2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cδ (PKCδ) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCδ activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 α2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCδ signaling pathway.     

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser⁷⁷⁹ in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser⁷⁷⁹ in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser⁷⁷⁹ was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser⁷⁷⁹ in vitro, whereas overexpression of PKCϵ results in constitutive Ser⁷⁷⁹ phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser⁷⁷⁹ phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser⁷⁷⁹, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.     

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]_i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the I_Ca,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser¹⁶ was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca²⁺ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.     

Phosphorylation of Serine 399 in LKB1 Protein Short Form by Protein Kinase Cζ Is Required for Its Nucleocytoplasmic Transport and Consequent AMP-activated Protein Kinase (AMPK) Activation

Two splice variants of LKB1 exist: LKB1 long form (LKB1_L) and LKB1 short form (LKB1_S). In a previous study, we demonstrated that phosphorylation of Ser-428/431 (in LKB1_L) by protein kinase Cζ (PKCζ) was essential for LKB1-mediated activation of AMP-activated protein kinase (AMPK) in response to oxidants or metformin. Paradoxically, LKB1_S also activates AMPK although it lacks Ser-428/431. Thus, we hypothesized that LKB1_S contained additional phosphorylation sites important in AMPK activation. Truncation analysis and site-directed mutagenesis were used to identify putative PKCζ phosphorylation sites in LKB1_S. Substitution of Ser-399 to alanine did not alter the activity of LKB1_S, but abolished peroxynitrite- and metformin-induced activation of AMPK. Furthermore, the phosphomimetic mutation (S399D) increased the phosphorylation of AMPK and its downstream target phospho-acetyl-coenzyme A carboxylase (ACC). PKCζ-dependent phosphorylation of Ser-399 triggered nucleocytoplasmic translocation of LKB1_S in response to metformin or peroxynitrite treatment. This effect was ablated by pharmacological and genetic inhibition of PKCζ, by inhibition of CRM1 activity and by substituting Ser-399 with alanine (S399A). Overexpression of PKCζ up-regulated metformin-mediated phosphorylation of both AMPK (Thr-172) and ACC (Ser-79), but the effect was ablated in the S399A mutant. We conclude that, similar to Ser-428/431 (in LKB1_L), Ser-399 (in LKB1_S) is a PKCζ-dependent phosphorylation site essential for nucleocytoplasmic export of LKB1_S and consequent AMPK activation.     

Serine 129 Phosphorylation Reduces the Ability of ��-Synuclein to Regulate Tyrosine Hydroxylase and Protein Phosphatase 2A in Vitro and in Vivo

α-Synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.     

Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain ��G-Helix

AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain αG-helix. Mutation of the corresponding AMPK α-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the αG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (α1^−/−, α2^−/−) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the αG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Cα was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic αG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic αG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.The maintenance of energy homeostasis is a basic requirement of all living organisms. The AMP-activated protein kinase (AMPK)² is crucially involved in this essential process by playing a central role in sensing and regulating energy metabolism on the cellular and whole body level (1–6). AMPK is also participating in several signaling pathways associated with cancer and metabolic diseases, like type 2 diabetes mellitus, obesity, and other metabolic disorders (7–9).Mammalian AMPK belongs to a highly conserved family of serine/threonine protein kinases with homologs found in all eukaryotic organisms examined (1, 3, 10). Its heterotrimeric structure includes a catalytic α-subunit and regulatory β- and γ-subunits. These subunits exist in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) and splice variants (for γ2 and γ3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The α- and β-subunits possess multiple autophosphorylation sites, which have been implicated in regulation of subcellular localization and kinase activation (11–15). The most critical step of AMPK activation, however, is phosphorylation of Thr-172 within the activation segment of the α-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been identified so far, namely the tumor suppressor kinase LKB1 in complex with MO25 and STRAD (16) and Ca²⁺/calmodulin-dependent protein kinase kinase-2 (CamKK2) (17). Furthermore, the transforming growth factor-β-activated kinase 1 was also shown to activate AMPK using a variety of in vitro approaches (18), but the physiological relevance of these findings remains unclear. Besides direct phosphorylation of Thr-172, AMPK activity is stimulated by the allosteric activator AMP, which can bind to two Bateman domains formed by two pairs of CBS domains within the γ-subunit (19–22). Hereby bound AMP not only allosterically stimulates AMPK but also protects Thr-172 from dephosphorylation by protein phosphatase 2Cα (PP2Cα) and thus hinders inactivation of the kinase (19, 22, 23). Consequently, on the cellular level, AMPK is activated upon metabolic stress increasing the AMP/ATP ratio. Furthermore, AMPK activation can also be induced by several chemical compounds, like nucleoside 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (24) and the anti-diabetic drug Metformin (25–28). In addition, the small molecule compound A-769662 was recently developed as a direct allosteric activator of AMPK (29, 30).Previous work in our groups proposed a model of AMPK regulation by AMP, which incorporates the major functional features and the latest structural information (31). The latter mainly included truncated core complexes of AMPK from different species (32–35). Further valuable structural information is provided by the x-ray structures of the isolated catalytic domains, in particular of the human AMPK α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog SNF1 (36, 37). The kinase domain of SNF1 is capable of forming homodimers in the protein crystal, as well as in vitro in solution, in a unique way, which has not been observed previously in any other kinase (36). The dimer interface is predominantly formed by hydrophobic interactions of the loop-αG region, also known as subdomain X situated on the large kinase lobe (36, 38, 39), and it mainly involves Ile-257 and Phe-261. Because the T-loop activation segment was buried within the dimer interface, it was suggested that the dimeric state of the SNF1 catalytic domain represents the inactive form of the kinase. Intriguingly, it was shown in our groups by small angle x-ray scattering that AMPK self-organizes in a concentration-dependent manner to form homo-oligomers in solution (31). However, the interface responsible for oligomerization of the AMPK heterotrimer has remained elusive.Here we further investigate the distinct oligomeric states of the AMPK heterotrimer and suggest a possible regulatory function for this process. Most importantly, we provide conclusive evidence for participation of αG-helix residues in the recognition of AMPK by its upstream kinases LKB1 and CamKK2.