PKB/Akt

The PDK1–PKB/Akt axis represents one of the most actively researched cell signaling pathways. This protein kinase cascade is known to play a central role in mediating the actions of a range of stimuli including insulin, growth factors, integrins and GPCRs in addition to being involved in the regulation of cell survival, cellular metabolism (including insulin-stimulated glucose transport and glycogen synthesis), gene expression, cell cycle entry and protein synthesis.

All the kinases associated with this pathway lie in the protein serine/threonine kinase family and form a single highly branching protein kinase cascade (hence their grouping together). Several of these kinases contain pleckstrin homology (PH) domains that bind specific phosphoinositide lipids (e.g. phosphoinositide-3,4,5-trisphosphate; PIP₃) which are generated in the plasma membrane in response to agonist activity. As a result, the kinases are activated in a phosphoinositide 3-OH-kinase (PI3-kinase)- dependent manner.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) stands at the head of this important signaling pathway. Whether extracellular stimuli directly activate PDK1 (perhaps via the generation of plasma membrane-localized PIP₃), or whether they simply induce the translocation of PDK1 to its substrate proteins within the plasma membrane, is not known. PDK1 activates a number of AGC-family protein kinases (named after their homology to protein kinases A, G and C) by phosphorylation, including protein kinase B (PKB or Akt) via phosphorylation of the T-loop residue Thr 308. The full activation of PKB/Akt also involves the binding of PIP₃ to the PH domain of PKB/Akt and the phosphorylation of an additional residue, Ser 473, by an as yet unidentified kinase called “PDK2” (proposed to be DNA-dependent protein kinase: DNA-PK). There is a great deal of functional overlap between PKB/Akt isoforms; all phosphorylate the same RXRXXS/T motif and all are capable of transforming a cell when rendered constitutively active by the introduction of a myristoylation signal sequence.

Thr 308 and Ser 473 lie within regions of PKB/Akt that are conserved throughout the AGC family kinases. Hence, PDK1 also phosphorylates and activates several other AGC-family kinases, including the serum and glucocorticoid-induced kinases (SGK), atypical forms of protein kinase C (e.g. PKCζ and PKCι/λ), p70S6-kinase and p90RSK. PDK1 is therefore a central controller of multiple cell signaling pathways.

Once phosphorylated and activated, PKB/Akt phosphorylates and inhibits glycogen synthase kinase 3 (GSK3) leading to a decreased phosphorylation and activation of glycogen synthase. PKB/Akt also phosphorylates the mammalian target of rapamycin (mTOR, also known as FRAP and RAFT) although the role of this action is not yet known. GSK3 continues to grow in importance as it also plays a role in the regulation of β-catenin stability and thus gene expression.

mTOR, which can be phosphorylated by PKB/Akt, is unusual in that it possesses both serine/threonine protein kinase as well as lipid kinase activities. It is a large complex molecule that is a receptor for the immunosuppressant, rapamycin. mTOR, along with PDK1, then plays an as yet ill-defined role in the activation of p70S6K which is important in the control of protein synthesis, development and growth control. Thus, at least in part, the immunosuppressive activity of rapamycin is due to its actions on mTOR.

Other than rapamycin, there are few if any highly specific pharmacological inhibitors of this collection of protein kinases. This has made understanding the role of these protein kinases in mediating the effects of extracellular stimuli very difficult to ascertain. Furthermore, while in some cases kinase-dead derivatives of these kinases have been reported to act as dominant-negatives, this has often been highly controversial, largely because of their complex domain structures and abilities to interact with other proteins and signaling lipids. However, given the central importance of these protein kinases in numerous disease states (e.g. cancer and diabetes), the identification of specific inhibitors remains a very important goal.

Family Members	PDK1	PKB/Akt	SGK
Other Names	3-Phosphoinositide protein kinase-1	RAC-protein kinase	Serum and glucacorticoid-induced kinase CISK (SGK3)
Molecular Weight (kDa)	63 kDa	56 kDa (All isoforms)	SGK1: 49 kDa SGK2α: 48 kDa SGK2β: 41 kDa SGK3 57: kDa
Structural Data	556 aa Monomer	PKBα: 480 aa PKBβ: 481 aa PKBγ: 479 aa All monomers	SGK1: 431 aa SGK2α: 367 aa SGK2β: 427 aa SGK3: 496 aa All monomers
Isoforms	Not Known	PKBα/Akt1 PKBβ/Akt2 PKBγ/Akt3	SGK1 SGK2α/2β (splice variants) SGK3
Species	All eukaryotes	All metazoans (kinases orthologous to PKB and SGK exist in fungi, plasmodium, dictystelium)	All metazoans (kinases orthologous to PKB and SGK exist in fungi, plasmodium, dictystelium)
Domain Organization	1 PH domain binds PtdIns(3,4,5)P3	1 PH domain binds PtdIns(3,4,5)P3 PtdIns(3,4)P2	Protein kinase domain
Phosphorylation Sites	Ser241 (autophosphorylation)	PKBα sites, but conserved: Thr308 Ser473 Thr450 (constitutive) Tyr315 Tyr326	SGK1 sites, but conserved: Thr256 Ser442 Ser78
Tissue Distribution	Ubiquitous	Ubiquitous	Ubiquitous
Subcellular Localization	Cytosolic Membranes	Cytosolic Membranes	Cytosolic Membranes (SGK3)
Binding Partners/ Associated Proteins	Not Known	CTMP APPL Periplakin POSH HSP90 PKCθ Brk PKCζ IMPDH Ft1 TCL1	Not Known
Upstream Activators	Complex-see Overview	Phosphorylation by PDK1 and "PDK2" and binding of PIP3	Phosphorylation by PDK1 (SGK1 expression induced by glucocorticoids)
Downstream Activation	PKA (P5511, C8482, P2645) PKG PKB/Akts MSKs atypical PKCs p70S6Ks p90RSKs (all are protein kinases)	GSK3 mTOR Raf IKK BAD eNOS hCaspase-9 (C1099) hTERT BRCA1 IRS-1 PFK2 FKHR PRAS40 TSC2 AS160 PIKfyve hdm-2 WNK1	FKHR NDRG1 NDRG2 Nedd4-2 B-Raf
Activators	Not Known	Not Known	Not Known
Inhibitors	UCN-01 (U6508)	SH5 API-2 (T3830) AKT-I-1 Akt-I-1.2	Not Known
Selective Activators	Not Known	Not Known	Not Known
Physiological Function	Cell proliferation Growth Apoptosis Metabolism	Cell proliferation Growth Apoptosis Metabolism	Regulation of Na+ and Cl- channels
Disease Relevance	Cancer Inflammation Others	Cancer Inflammation Others	Hypertension

Family Members	GSK3	mTOR	p70S6K (S6K)
Other Names	Glycogen synthase kinase 3 (G1663)	FRAP RAFT Mammalian target of rapamycin	S6K1
Molecular Weight (kDa)	GSK3α: 51 kDa GSK3β: 48 kDa	289 kDa	α1: 59 kDa α2: 56 kDa β1: 55 kDa β2: 54 kDa
Structural Data	483 aa β: 420 aa All monomers	2549 aa Monomer	α1: 525 aa α2: 502 aa β1: 495 aa β2: 482 aa All monomers
Isoforms	GSK3α GSK3β	Not Known	p85 S6Kα1 = α1 p70 S6Kα2 = α2 (splice variants of the S6K1 gene) p60 S6Kβ1 = β1 p54 S6Kβ2 = β2 (splice variants of the S6K2 gene)
Species	All eukaryotes	All eukaryotes	All metazoans
Domain Organization	Not Known	16 HEAT domains 1 PI3-kinase homology domain 1 FKBP/rapamycin binding domain	1 autoinhibitory domain 1 nuclear localization signal sequence on α1, β1 and β2
Phosphorylation Sites	GSK3α, but conserved in GSK3β: Ser21 (PKB and others) Tyr296 (constitutive)	Ser2448 Ser2481 (autophosphorylation)	Sites α2, but conserved: Thr229 Ser371 Ser389 Ser411 Ser418 Thr424 In α2, conserved in α1 only: Thr421
Tissue Distribution	Ubiquitous	Ubiquitous	Ubiquitous
Subcellular Localization	Cytosolic Nuclear	Cytosolic	Cytosolic (α2) Nuclear (α1, β1 and β2)
Binding Partners/ Associated Proteins	Frat-1/2/3 Presenilin Axin Axil DLP 14-3-3	FKBP12 Raptor Rictor Rheb LST8	PP2A Neurabin
Upstream Activators	Tyrosine phosphorylation (constitutive) Phosphorylation by PKB/Akt	Complex, but may include phosphorylation by PKB and regulation by TSC2/Rheb	Phosphorylation by PDK1, mTOR and atypical PKCs
Downstream Activation	Glycogen synthase ATP-citrate lyase eIF-2B c-Jun Myc Myb PKA (P5511, C8482, P2645) CREB IkB PP1 (P7937) tau NDRG1	4E-BP1 p70S6K	Ribosomal S6 subunit SKAR
Activators	Not Known	Not Known	Not Known
Inhibitors	SB-216763 (S3442) SB-415286 (S3567) Lithium Chloride (213233) CT99021 AR-A014418 (A3230)	Rapamycin (R0395)	H89 (B1427)
Selective Activators	Not Known	Not Known	Not Known
Physiological Function	Glycogen metabolism Gene expression Development	Protein synthesis Cell growth	Protein synthesis Cell growth
Disease Relevance	Diabetes Metabolism	Cancer Immunosuppression	Cancer

Footnotes

a) Inhibitors in parentheses are non-selective or yet unproven to be highly specific.

Abbreviations

Brk: Breast tumor-related kinase
CTMP: C-terminal modulatory protein
DLP: Dynamin-like protein
DNA-PK: DNA-dependent protein kinase
FKBP12: FK502 binding protein
FRAP: FKBP12-rapamycin-associated protein
GSK3: Glycogen synthase kinase 3
H89: N-(2-[p-Bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide
IMPDH: Inosine-5’ monophosphate dehydrogenase
MSK: Mitogen- and stress-activated kinase
MTOR: Mammalian target of rapamycin
PIKfyve: FYVE domain containing phosphatidylinositol 3-phosphate 5-kinase
PKA: Protein kinase A
PKC: Protein Kinase C
PKG: Protein kinase G
POSH: Plenty of SH3 domains
PtdIns(3,4)P2: Phosphatidylinositol 3,4-bisphosphate
PtdIns(3,4,5)P3: Phosphatidylinositol 3,4,5-trisphosphate
RAFT: Rapamycin and FKBP12 target
Ro 31-8220: 2-{1-[3-Amidinothio)propyl]-1H-indol-3-yl}-3-(1-methylindol-3-yl)-maleimide
SB-216763: 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indole-3-yl)-1H-pyrrole-2,5-dione
SB-415286: 3-(3-Chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione
SGK: Serum and glucocorticoid regulated kinase
TCL: T-cell leukemia
TSC2: Tuberous Sclerosis Complex-2
UCN-1: 7-Hydroxystaurosporine