mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1)

2008 ◽  
Vol 416 (3) ◽  
pp. 375-385 ◽  
Author(s):  
Juan M. García-Martínez ◽  
Dario R. Alessi
Keyword(s):  
Protein Kinase ◽  
Mammalian Target Of Rapamycin ◽  
Kinase Domain ◽  
Vital Role ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Kinase C ◽  
Forkhead Box ◽  
Hydrophobic Motif ◽  
Mcf 7

SGK1 (serum- and glucocorticoid-induced protein kinase 1) is a member of the AGC (protein kinase A/protein kinase G/protein kinase C) family of protein kinases and is activated by agonists including growth factors. SGK1 regulates diverse effects of extracellular agonists by phosphorylating regulatory proteins that control cellular processes such as ion transport and growth. Like other AGC family kinases, activation of SGK1 is triggered by phosphorylation of a threonine residue within the T-loop of the kinase domain and a serine residue lying within the C-terminal hydrophobic motif (Ser422 in SGK1). PDK1 (phosphoinositide-dependent kinase 1) phosphorylates the T-loop of SGK1. The identity of the hydrophobic motif kinase is unclear. Recent work has established that mTORC1 [mTOR (mammalian target of rapamycin) complex 1] phosphorylates the hydrophobic motif of S6K (S6 kinase), whereas mTORC2 (mTOR complex 2) phosphorylates the hydrophobic motif of Akt (also known as protein kinase B). In the present study we demonstrate that SGK1 hydrophobic motif phosphorylation and activity is ablated in knockout fibroblasts possessing mTORC1 activity, but lacking the mTORC2 subunits rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated-protein-kinase-interacting protein 1) or mLST8 (mammalian lethal with SEC13 protein 8). Furthermore, phosphorylation of NDRG1 (N-myc downstream regulated gene 1), a physiological substrate of SGK1, was also abolished in rictor-, Sin1- or mLST8-deficient fibroblasts. mTORC2 immunoprecipitated from wild-type, but not from mLST8- or rictor-knockout cells, phosphorylated SGK1 at Ser422. Consistent with mTORC1 not regulating SGK1, immunoprecipitated mTORC1 failed to phosphorylate SGK1 at Ser422, under conditions which it phosphorylated the hydrophobic motif of S6K. Moreover, rapamycin treatment of HEK (human embryonic kidney)-293, MCF-7 or HeLa cells suppressed phosphorylation of S6K, without affecting SGK1 phosphorylation or activation. The findings of the present study indicate that mTORC2, but not mTORC1, plays a vital role in controlling the hydrophobic motif phosphorylation and activity of SGK1. Our findings may explain why in previous studies phosphorylation of substrates, such as FOXO (forkhead box O), that could be regulated by SGK, are reduced in mTORC2-deficient cells. The results of the present study indicate that NDRG1 phosphorylation represents an excellent biomarker for mTORC2 activity.

Janus Kinases Interact with and Phosphorylate Rack-1 (Receptor for Activated C Kinase-1), a WD Motif Containing Protein.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2184-2184
Author(s):  
Takashi Haro ◽  
Kazuya Shimoda ◽  
Haruko Kakumitsu ◽  
Kenjirou Kamezaki ◽  
Atsuhiko Numata ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
B Lymphocyte ◽  
Kinase Domain ◽  
Scaffolding Protein ◽  
Cytokine Signaling ◽  
Interacting Protein ◽  
Kinase C ◽  
Tyrosine Kinase 2 ◽  
Lymphocyte Growth

Abstract We recently reported that Tyk2 was essential for IFN-a-induced B lymphocyte growth inhibition, although Stat1 is not required for this IFN-a-mediated inhibition. This means that other signaling molecules besides Stat1, and which are activated by Tyk2, are thought to transduce the IFN-a signal inhibiting B lymphocyte growth. We performed a yeast two-hybrid screen for proteins that interact with Tyk2, and identified Rack-1, originally described as a receptor for activated C kinase beta, associated with Tyk2. Receptor for activated C kinase (Rack)-1 is a protein kinase C interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase C gamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alpha/beta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudo-kinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N-terminus and one in the middle portion (a.a.138–203) of Rack-1. In addition, not only Tyk2 but other Jak kinases associate with Rack-1, and each Jak activation causes the phosphorylation of Tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated from cytoplasm or membrane toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.

mTORC2 is the hydrophobic motif kinase for SGK1

2008 ◽  
Vol 416 (3) ◽  
pp. e19-e21 ◽  
Author(s):  
Lijun Yan ◽  
Virginie Mieulet ◽  
Richard F. Lamb
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Kinase C ◽  
Biochemical Journal ◽  
Dependent Protein ◽  
Hydrophobic Motif ◽  
Phosphoinositide 3 Kinase ◽  
A Site

The activation of the AGC (protein kinase A/protein kinase G/protein kinase C)-family kinase SGK1 (serum- and glucocorticoid-induced kinase 1) by insulin via PI3K (phosphoinositide 3-kinase) signalling has been appreciated for almost 10 years. PDK1 (phosphoinositide-dependent protein kinase 1), a kinase that phosphorylates the SGK1 catalytic domain at Thr256, is known to play a critical role in SGK1 activation. However, the identity of the protein kinase(s) responsible for phosphorylation of Ser422, a site outside the catalytic domain (the so-called hydrophobic motif, or HM) that promotes activation of the kinase by PDK1, was unclear. In work reported in this issue of the Biochemical Journal, García-Martínez and Alessi have revealed the identity of a ‘PDK2’ kinase that catalyses Ser422 phosphorylation as mTORC2 (mammalian target of rapamycin complex 2), a multiprotein kinase that phosphorylates a similar site in PKB (protein kinase B).

Regulation of novel protein kinase C ε by phosphorylation

2002 ◽  
Vol 363 (3) ◽  
pp. 537-545 ◽  
Author(s):  
Vittoria CENNI ◽  
Heike DÖPPLER ◽  
Erica D. SONNENBURG ◽  
Nadir MARALDI ◽  
Alexandra C. NEWTON ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
Kinase Domain ◽  
Activation Loop ◽  
3T3 Fibroblasts ◽  
Kinase C ◽  
Hydrophobic Motif ◽  
Nih 3T3

The activity and intracellular localization of protein kinase C (PKC) family members are controlled by phosphorylation at three highly conserved sites in the catalytic kinase domain. In the case of the novel PKC∊ isoform, these are Thr566 in the activation loop, Thr710 in the turn motif and Ser729 in the C-terminal hydrophobic motif. In the present study, we analysed the contribution of the phosphoinositide-dependent kinase 1 (PDK-1) and PKC∊ kinase activity in controlling the phosphorylation of Thr566 and Ser729. In NIH 3T3 fibroblasts, PKC∊ migrated as a single band, and stimulation with platelet-derived growth factor resulted in the appearance of a second band with a slower electrophoretic mobility, concomitant with an increase in phosphorylation of Thr566 and Ser729. Cells transfected with an active PDK-1 allele also resulted in increased PKC∊ Thr566 and Ser729 phosphorylation, whereas an active myristoylated PKC∊ mutant was constitutively phosphorylated at these sites. Protein kinase-inactive mutants of PKC∊ were not phosphorylated at Ser729 in cells, and phosphorylation of this site leads to dephosphorylation of the activation-loop Thr566, an effect which can be reversed with either okadaic acid or co-transfection with active PDK-1. In vitro, PDK-1 catalysed the phosphorylation of purified PKC∊ in the presence of mixed micelles containing either diacylglycerol or PtdIns(3,4,5)P3, concomitant with an increase in Ser729 phosphorylation. These studies reveal that the mechanism of phosphorylation of a novel PKC is the same as that for conventional PKCs: PDK-1 phosphorylation of the activation loop triggers autophosphorylation of the hydrophobic motif. However, the regulation of this phosphorylation is different for novel and conventional PKCs. Specifically, the phosphorylation of novel PKCs is regulated rather than constitutive.

Early signalling events of autophagy

2013 ◽  
Vol 55 ◽  
pp. 1-15 ◽  
Author(s):  
Laura E. Gallagher ◽  
Edmond Y.W. Chan
Keyword(s):  
Protein Kinase ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Mammalian Cells ◽  
Mammalian Target Of Rapamycin ◽  
Interacting Protein ◽  
The Core ◽  
Autophagy Gene ◽  
Autophagy Induction ◽  
Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

RGSZ1 interacts with protein kinase C interacting protein PKCI-1 and modulates mu opioid receptor signaling

2007 ◽  
Vol 19 (4) ◽  
pp. 723-730 ◽  
Author(s):  
Seena K. Ajit ◽  
Suneela Ramineni ◽  
Wade Edris ◽  
Rachel A. Hunt ◽  
Wah-Tung Hum ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Opioid Receptor ◽  
Mu Opioid Receptor ◽  
Receptor Signaling ◽  
Interacting Protein ◽  
Mu Opioid ◽  
Kinase C

scholarly journals Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

1999 ◽  
Vol 144 (3) ◽  
pp. 403-411 ◽  
Author(s):  
Shun'ichi Kuroda ◽  
Noritaka Nakagawa ◽  
Chiharu Tokunaga ◽  
Kenji Tatematsu ◽  
Katsuyuki Tanizawa
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Caenorhabditis Elegans ◽  
Protein Kinase ◽  
Rat Brain ◽  
Axonal Outgrowth ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Kinase C ◽  
Two Hybrid

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.

scholarly journals Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR)

2009 ◽  
Vol 421 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Juan M. García-Martínez ◽  
Jennifer Moran ◽  
Rosemary G. Clarke ◽  
Alex Gray ◽  
Sabina C. Cosulich ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Mammalian Target Of Rapamycin ◽  
Specific Inhibitor ◽  
Initiation Factor ◽  
Target Of Rapamycin ◽  
S6 Kinase ◽  
Class 1 ◽  
Hydrophobic Motif ◽  
Ribosomal S6 Kinase

mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of ∼10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

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