Báo cáo khoa học: G protein-coupled receptor-induced Akt activity in cellular proliferation and apoptosis pptx

PI3K pathways are activated in response to extracellular signals medi-ated by cell-surface receptors of the G protein-coupled receptor GPCR, integrin and growth factor⁄ receptor tyrosine

G protein-coupled receptor-induced Akt activity in cellular proliferation and apoptosis

David C New, Kelvin Wu, Alice W S Kwok and Yung H Wong

Department of Biochemistry, the Molecular Neuroscience Center, and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

Akt, also known as protein kinase B (PKB), is a

ser-ine⁄ threonine protein kinase that plays a pivotal role

in many physiological processes, including metabolism,

development, cell cycle progression, migration and

sur-vival [1–4] The Akt subfamily of protein kinases

con-sists of three isoforms – Akt1, Akt2 and Akt3 (also

termed PKBa, PKBb and PKBc) – which are the

products of distinct genes All three proteins share a

conserved tertiary structure of an N-terminal pleckstrin

homology domain, a kinase domain and a C-terminal

regulatory domain containing the hydrophobic motif

phosphorylation site [5] While the homology between

the three isoforms allows for a degree of functional

redundancy [1], there also seems to be considerable

scope for isoform-specific activation and substrate specificity [3,6]

Akt plays an integral role in the phosphoinositide 3-kinase (PI3K) signaling pathways PI3K pathways are activated in response to extracellular signals medi-ated by cell-surface receptors of the G protein-coupled receptor (GPCR), integrin and growth factor⁄ receptor tyrosine kinase (RTK) superfamilies Receptor-medi-ated activation of PI3K results in the generation of phosphatidylinositol (3,4,5)-trisphosphate from phos-phatidylinositol (4,5)-bisphosphate, a reaction that is reversed by the enzymes phosphatase and tensin homo-logue (PTEN) and SH2-domain-containing inositol polyphosphate 5-phosphatase (SHIP) Both Akt and

Keywords

Akt; protein kinase B; G protein; G

protein-coupled receptor; cell cycle; apoptosis

Correspondence

Y H Wong, Department of Biochemistry,

Hong Kong University of Science and

Technology, Clear Water Bay, Kowloon,

Hong Kong, China

Fax: +852 2358 1552

Tel: +852 2358 7328

E-mail: boyung@ust.hk

(Received 17 August 2007, revised 17

Sep-tember 2007, accepted 24 SepSep-tember 2007)

doi:10.1111/j.1742-4658.2007.06116.x

Akt (also known as protein kinase B) plays an integral role in many intra-cellular signaling pathways activated by a diverse array of extraintra-cellular sig-nals that target several different classes of membrane-bound receptors Akt plays a particularly prominent part in signaling networks that result in the modulation of cellular proliferation, apoptosis and survival Thus, the over-expression of Akt subtypes has been measured in a number of cancer types, and dominant-negative forms of Akt can trigger apoptosis and reduce the survival of cancer cells G protein-coupled receptors act as cell-surface detectors for a diverse spectrum of biological signals and are able to acti-vate or inhibit Akt via several direct and indirect means In this review, we shall document how G protein-coupled receptors are able to control Akt activity and examine the resulting biochemical and physiological changes, with particular emphasis on cellular proliferation, apoptosis and survival

Abbreviations

CDK, cyclin-dependent kinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FH, forkhead; GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NF-jB, nuclear factor jB; p70 S6K , p70 ribosomal protein S6 kinase; PAR-2, protease-activated receptor-2; PDGFRa, platelet-derived growth factor receptor a; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; PTEN, phosphatase and tensin homologue; RTK, receptor tyrosine kinase; SHIP, SH2-domain-containing inositol polyphosphate 5-phosphatase; TNF, tumour necrosis factor; TSC, tuberous sclerosis complex; TSH, thyroid-stimulating hormone receptor.

phosphoinositide-dependent kinase are recruited to the

plasma membrane by phosphatidylinositol

(3,4,5)-tris-phosphate through their pleckstrin homology domains,

where phosphoinositide-dependent kinase

phosphory-lates Akt1 on residue Thr308 in its kinase domain [7]

A second phosphorylation takes place at Ser473 in the

hydrophobic motif region of Akt1 This

phosphoryla-tion event seems to be catalyzed by a number of

differ-ent kinases, which are probably stimulus- and⁄ or cell

type-specific This stabilizes the active conformation of

Akt and allows it to translocate to the cytoplasm or

nucleus to search for its many target proteins [8]

Akt’s role in physiology suggests that aberrant Akt

signaling may be a factor in disease states Most

nota-bly, amplification of Akt isoform genes and Akt

mRNA overexpression has been observed in many

human cancers [9] Akt activity in cancer cells may

also be enhanced by the amplification of genes

encod-ing PI3K or by a reduction in the activity of PTEN or

SHIP [9] It is therefore to be expected that the

inhibi-tion of PI3K, Akt and their downstream effectors has

been targeted in the development of cancer therapies

[10] The involvement of Akt in cancer is not

surpris-ing given the ability of Akt to promote cellular

proliferation through the direct and indirect

phosphor-ylation of a number of cell cycle regulatory proteins

[11], and its ability to inactivate pro-apoptotic factors,

such as Bad, caspase-9 and forkhead (FH)

transcrip-tion factors [12] In contrast, it is thought that a

reduc-tion in Akt signaling may contribute to diabetes by

reducing the survival of pancreatic b cells [13]

GPCRs act as cell-surface detectors for

a diverse spectrum of biological signals

To date, over 200 GPCRs have been matched with a

ligand that activates the receptor to promote a wide

variety of intracellular biochemical changes [14], even

though it is estimated that the human genome encodes

between 800 and 1000 GPCR subtypes [15,16] Their

pervasive influence, coupled with their cell-surface

accessibility, has resulted in GPCRs becoming the

tar-gets of as many as 45% of modern medicines [17],

which are used to treat conditions as diverse as

inflam-mation, incontinence, hypertension, depression and

pain [18]

GPCRs preferentially couple to heterotrimeric

G proteins (consisting of a, b and c subunits) that are

grouped into four classes, known as Gaq ⁄ 11, Gai ⁄ o, Gas

and Ga12 ⁄ 13, based on the effector with which the

a-subunit primarily interacts The activated G proteins

in turn promote the activation or inhibition of a variety

of intracellular events, including the activation of

phos-pholipases, mitogen-activated protein kinases (MAP-Ks), activation⁄ inhibition of adenylyl and guanylyl cyclases, and the opening and closing of ion channels

In this review, we shall investigate the ability of GPCRs to activate Akt signaling pathways both directly, through the interaction of Gbc subunits with PI3K, and indirectly, through the GPCR transactiva-tion of RTKs and integrins We shall also examine the downstream signaling and physiological consequences

of GPCR-induced Akt activation, paying particular attention to the consequences for cellular proliferation, survival and apoptosis

pathways

GPCRs promote intracellular signaling through both

Ga and Gbc subunits, which can activate distinct, complementary or antagonistic pathways As we will demonstrate, a large number of GPCRs, coupled to all four classes of G protein, activate PI3K⁄ Akt pathways through either Ga or Gbc subunits (see below, Table 1 and Fig 1) Gbc subunits are able to bind directly to and activate PI3K heterodimeric proteins containing either the p110b or the p110c subunits [19] Further-more, it has been demonstrated that muscarinic and lysophosphatidic acid (LPA) GPCRs are only able to activate Akt in cells expressing the p110b or p110c subunits, and that this activation is mediated by Gbc subunits, but not by Ga subunits [20] Direct activa-tion of PI3K by Ga subunits has not been specifically measured but they can activate PI3K⁄ Akt pathways by transactivating integrins, RTKs and other growth fac-tor recepfac-tors

Numerous RTKs and integrins can independently activate PI3K⁄ Akt pathways [21] but it has also been reported that they can be transactivated by GPCRs through Ga- or Gbc-dependent pathways [22,23] For example, ligands for the LPA, endothelin-1 and throm-bin receptors all promote DNA synthesis in Rat1 fibroblasts by transactivating the epidermal growth factor receptor (EGFR, an RTK) Such transactivation requires the activation of matrix metalloproteases to release EGF from its membrane-bound form, which then stimulates the EGFR and downstream extracellu-lar signal-regulated kinase (ERK) pathways [24] PI3K⁄ Akt pathways are also activated by a similar method of transactivation [25] In Swiss 3T3 cells, bradykinin and bombesin promote cellular prolifera-tion by an EGFR-dependent formaprolifera-tion of a signaling complex that activates PI3K⁄ Akt cascades [26] A number of other RTKs are also transactivated by GPCRs [27–29], and it has been demonstrated that

Table 1 GPCR-induced Akt activity and the consequences for cellular proliferation and apoptosis A selection of examples is presented here demonstrating the signaling components employed in connecting GPCR-initiated signals to downstream events regulated by Akt ›, indicates

an increase in protein levels or activity; fl, indicates a decrease in protein levels or activity AC, adenylyl cyclase; AFX, FOX04; ALXR, lipoxin

A4receptor; CDK, cyclin-dependent kinase; CREB, cAMP-response element binding; cyt c, cytochrome c; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FKHR, forkhead in rhabdomyosarcoma; FSH, follicle stimulating hormone; IGF-1, insulin growth factor-1; IGF-IRb, insulin-like growth factor receptor; IRS-1, insulin receptor substrate 1; GnRH, gonadotropin-releasing hormone; GSK3b, glycogen synthase kinase 3b; LHRH, luteinizing hormone-releasing hormone; LPA, lysophophatidic acid; MMP, matrix metalloprotein-ase; mTOR, mammalian target of rapamycin; NF-jB, nuclear factor-jB; p70 S6k , p70 ribosomal protein S6 kinase; PAR-2, protease-activated receptor-2; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PP2A, protein phosphatase 2A; PTP, protein tyro-sine phosphatase, ROCK, Rho-associated kinase; ROS, reactive oxygen species; SHP, Src homology 2-containing phosphatase; TSC2, tuber-ous sclerosis complex 2; TSH, thyroid stimulating hormone; VPAC, vasoactive intestinal peptide receptor.

Gi⁄ opathways

ALXR flPDGF ⁄ ›EGF ⁄ ›PI3K ⁄ ›Akt ⁄ ›p27 Kip1 ⁄ ›p21 Cip1 ⁄

flCDK2 ⁄ flcyclin E

Anti-inflammatory effects, antiproliferation

[47]

responses

[37]

proliferation, survival

[24,25]

antiangiogenic and anti-invasive

[36]

›Src ⁄ ›SHP-1 ⁄ ›SHP-2 ⁄ ›PI3K ⁄ ›Ras ⁄ ›ERK ⁄

›p27 Kip1

proliferation

[45]

Gq⁄ 11pathways

›EGFR ⁄ ›PI3K ⁄ ›Akt ⁄ ›mTOR ⁄ ›p70 S6K ›G1 cyclins, flp27 Kip1 , flp21 Cip1 ,

proliferation

[52]

Apelin APJ ›PI3K ⁄ ›Akt ⁄ flcaspase 8 ⁄ flcyt c ⁄ flcaspase 9 ⁄

flcaspase 3

synthesis, proliferation

[26]

›PTP ⁄ flphosphorylated-IRS-1 ⁄ flPI3K ⁄ flAkt ⁄ ›RhoA ⁄ flROCK-I

Table 1 (Continued).

Muscarinic subtypes ›Src ⁄ ›PI3K ⁄ ›Akt ⁄ ›ERK ⁄ flGSK3b ⁄ flcaspase 3 ⁄

›CREB

Survival, proliferation [67]

PAR-2 ›Ca 2+ ⁄ ›PKC ⁄ ›Pyk2 ⁄ ›Src ⁄ ›PI3K ⁄ ›Akt Actin reorganization and cell

migration

[33]

Vasopressin V 1 ›Ca 2+ ⁄ ›PKC ⁄ ›Pyk2 ⁄ ›Src ⁄ ›EGFR ⁄ ›PI3K ⁄ ›Akt ⁄

›mTOR ⁄ ›p70 S6K

Cell growth, proliferation [51]

Gspathways

development

[69]

TSH ›cAMP ⁄ ›PKA ⁄ ›PI3K ⁄ ›PDK1 ⁄ ›mTOR ⁄ ›p70 S6K

Proliferation, thyroid follicle activity

[54]

›cAMP ⁄ ›PKA ⁄ ›PI3K-Ras complex ⁄ flERK DNA synthesis, mitogenesis [55]

differentiation

[28]

G12⁄ 13 pathways

LPA ›Rho ⁄ ›p160ROCK ⁄ ›PDGFRa ⁄ ›PI3K ⁄ ›Akt ⁄ flFKHR flTranscription of apoptotic and

antiproliferative genes

[30] Thrombin ›Rho ⁄ ›p160ROCK ⁄ ›PDGFRa ⁄ ›PI3K ⁄ ›Akt ⁄ flFKHR flTranscription of apoptotic and

antiproliferative genes

[30]

Fig 1 Routes to Akt activation G12⁄ 13-, Gi⁄ o-, Gq-, and Gs-coupled receptors are all known to activate the phosphoinositide 3-kinase (PI3K) ⁄ Akt pathway through either Ga or Gbc subunits Ga q and Gassubunits utilize secondary messenger systems [Ca 2+ , cAMP, reactive oxygen species (ROS)] to promote PI3K ⁄ Akt activation Whilst Ca 2+

-induced PI3K activation is a feature of G i ⁄ o -coupled GPCRs, Gbc sub-units released from these receptors are also able to directly bind to and activate PI3K In addition to these mechanisms, all four classes of GPCRs are able to activate the PI3K ⁄ Akt pathway by transactivating RTK at the plasma membrane either through matrix metalloproteinases (G i ⁄ o -, G q -, and G s -coupled receptors) or through Rho ⁄ Rho-associated kinase (Rock)-mediated expression of RTK ligands (G 12 ⁄ 13 -coupled receptors) The Rho ⁄ Rock pathway can also indirectly inhibit PI3K activity, although the signaling components involved have not yet been elucidated (indicated by a dashed line).

constitutively active Ga12 subunits activate PI3K⁄ Akt

signaling via the transactivation of the platelet-derived

growth factor receptor a (PDGFRa) [30] However,

it is not clear whether transactivation of RTKs by

GPCRs can also occur through the induced expression

of RTK ligands Alternatively, the RTK ligand

requirement may be bypassed by the GPCR-induced

Src family tyrosine kinase activation of RTKs [27], as

evidenced by the Src kinase-dependent EGFR

transac-tivation promoted by the b-adrenergic receptor in

gas-tric mucosal cells [31]

Src-family kinases are firmly embedded in signal

transduction pathways activated by diverse

extracellu-lar stimuli playing a significant role in the crosstalk

between many pathways, including those that facilitate

the GPCR activation of Akt [32] The

protease-activated receptor-2 (PAR-2) utilizes Gaq subunits to

promote the activation of protein kinase C and the

mobilization of intracellular Ca2+, leading to the

formation of a complex containing Src-family kinases,

the focal adhesion kinase, Pyk2, and PI3K [33]

Similar findings have been made for the Gi⁄ o-coupled

CXCR4 receptor, which promotes DNA synthesis via

a Pyk2⁄ PI3K ⁄ ERK pathway [34] These complexes

may form as part of larger integrin⁄ paxillin signaling

platforms that promote the phosphorylation and

activation of PI3K subunits [35]

GPCR activation may also lead to the inactivation

of Akt It has been reported that somatostatin SST2

receptors directly form a complex with the p85

regula-tory subunit of PI3K Agonist activation induced the

dissociation of this complex, preventing PI3K

activa-tion [36] Following agonist-induced activaactiva-tion,

dopa-mine D2 receptors are internalized and form a

multiprotein complex that includes b-arrestin, protein

phosphatase 2A and Akt Protein phosphatase 2A

inactivates Akt, thereby relieving Akt’s inhibition of

glycogen synthase kinase 3b and allowing it to mediate

dopamine-induced neurological responses [37] An

alternative mode of b-arrestin-mediated PI3K⁄ Akt

inhibition is proposed to occur upon activation of the

Gaq-coupled PAR-2 receptor Upon recruitment to the

PAR-2 receptor, b-arrestin forms a complex with PI3K

and spatially restricts its enzyme activity, thereby

mod-ulating the PAR-2 receptor activation of PI3K⁄ Akt

mediated by Pyk2 and Src-family kinases (see the

pre-ceding discussion) [33] In contrast, another study has

indicated that b-arrestins mediate the endothelin A

receptor activation of Akt by recruiting Src-family

kin-ases that phosphorylate and activate Gaq, ultimately

leading to PI3K⁄ Akt pathway activation [38]

Never-theless, the vital role of b-arrestins in modulating the

apoptotic events following the activation of some

GPCRs was highlighted by a study which showed that

in mouse embryonic fibroblasts devoid of b-arrestins the N-formyl peptide receptor, vasopressin V2, chemo-kine CXCR2 and the angiotensin II AT1Areceptors all promote apoptosis through the activation of PI3K, MAPKs and Src kinases, leading to the activation of caspase pathways [39] Reconstituting the b-arrestins prevented the GPCR-induced apoptosis, suggesting that for some GPCRs b-arrestins constrain their apop-totic abilities The same study also demonstrated the GPCR selectivity of these events because in the absence of b-arrestins the CXCR4 and b2-adrenergic receptors were unable to activate apoptosis

Recent studies have indicated that constitutively active Ga subunits of the Gaq⁄ 11and Ga12⁄ 13 subfami-lies may actually inhibit the EGFR-mediated activa-tion of Akt in transfected HEK-293 cells [40] This contradicts the previously noted ability of constitutively active Ga12 subunits to potentiate PDGFRa-mediated PI3K⁄ Akt signaling [30] It is not immediately apparent whether these studies relate to GPCR signaling because RTKs are able to utilize heterotrimeric G protein pathways independently of GPCR activation [41]

Akt mediation of GPCR-induced cell cycle control

GPCRs have been widely reported to mediate mito-genic signals leading to cellular proliferation [2,42], and the overexpression or mutation of many GPCR subtypes in numerous cell types is thought to contrib-ute to deregulated growth and tumour development [43,44] The transmembrane and intracellular pathways mediating the GPCR control of cell cycle progression are extensive [2], with all pathways converging in the nucleus to regulate the expression, localization, activity

or stability of a small number of cell cycle proteins that are critical for the orderly progression from the G1 to S phases of the cell cycle Akt, in response to GPCR activation, directly interacts with some of these cell cycle proteins or exerts its effects through its downstream partners (Fig 2)

Evidence suggests that the GPCR activation of Akt pathways can be either proliferative or antiprolifera-tive, depending on the nature of the stimulus and the cell type observed Competing effects on cell cycle pro-gression generated simultaneously by the same extra-cellular signal have been observed, suggesting that the final outcome of a signaling event relies on the balance

of several competing mechanisms For example, activa-tion of the SST2a receptor in Chinese hamster ovary cells promotes the sustained activation of the MAPK

family member p38 and the up-regulation of the cell

cycle inhibitory protein p21Cip1 Conversely, activation

of the SST2b receptor resulted in the activation by

PI3K of both Akt and the p70 ribosomal protein S6

kinase (p70S6K), which led to cell cycle progression

[45], probably through induction of the expression of

cyclins (key proteins for the G1 to S phase transition)

[2] Both somatostatin receptor subtypes were shown

to be activating the same Gaisubtypes but it was

pos-tulated that the Gbc subunit pairings may have been

receptor subtype selective [45] Although we now know

that GPCR interactions with b-arrestins may also

con-trol PI3K⁄ Akt activation (as discussed above), a study

on a-thrombin receptor signaling demonstrated that

this GPCR activated Akt in b-arrestin-dependent and

-independent ways b-arrestin-independent activation

of Akt was more prolonged than b-arrestin-dependent

activation and led to cyclin D1 accumulation,

cy-clin D1-cycy-clin-dependent kinase (CDK) 4 activity and

cell cycle progression [46] The intermediaries between

Akt and cyclin D1 accumulation were not determined

but it is known that the cyclin D1 protein is stabilized

by the Akt-mediated inactivation of glycogen synthase

kinase 3b, which normally phosphorylates and

pro-motes the degradation of cyclin D1 In addition, Akt

also phosphorylates and inactivates FH transcription

factors, which bind to and activate the p27Kip1

pro-moter (another cell cycle inhibitory protein) Akt may

also reduce the stability of p27Kip1, and Akt

phosphor-ylation of p27Kip1adversely affects its nuclear

localiza-tion [11] Indeed, the anti-inflammatory lipoxins act

through GPCRs to inhibit the PDGFR-mediated acti-vation of Akt and the subsequent decrease in the levels

of p21Cip1 and p27Kip1, as well as inhibiting the PDGFR-mediated cyclin E–CDK2 complex formation and cell cycle progression [47]

Akt-induced phosphorylation of the tumour sup-pressor tuberous sclerosis complex (TSC)2 (also known

as tuberin) causes the dissociation of TSC2 and TSC1 (also known as hamartin), relieving their inhibition of the mammalian target of rapamycin (mTOR) kinase [48] Increased mTOR activity reduces the stability of p27Kip1, releasing its restrictions on cell cycle progres-sion In addition, mTOR activates the proliferative kinase p70S6K [11] Some GPCRs have now been shown to couple to this PI3K⁄ Akt ⁄ tuberin ⁄ mTOR sys-tem In PC-12 and other neuronal cells, the Gi⁄ o -cou-pled a2-adrenergic receptors, muscarinic M4 receptors,

as well as the d-, j- and l-opioid receptors, all pro-mote TSC2 phosphorylation via a PI3K⁄ Akt-depen-dent pathway [41,49,50] Despite such evidence, a direct role for a Gi-coupled GPCR⁄ Akt ⁄ mTOR signal-ing axis in cellular proliferation has not been demon-strated, as it has been for anti-apoptotic, pro-survival pathways (see below) However, activation of the Gq -coupled vasopressin V1 receptor in mesangial cells potently stimulates cell growth and proliferation by a Pyk2⁄ Src-dependent transactivation of EGFR followed

by an mTOR-dependent activation of p70S6K and cell cycle progression [51] A very similar proliferative EGFR⁄ PI3K ⁄ Akt ⁄ mTOR ⁄ p70S6Kpathway is activated

by Gq-coupled angiotensin II type 1 receptors in

Fig 2 Targets of Akt phosphorylation Acti-vated (phosphorylated) Akt isoforms are able to regulate key cellular and physiologi-cal processes by phosphorylating a wide range of substrates involved in cellular sur-vival (blue), glucose metabolism (orange), cell cycle progression (green), and protein synthesis (pink) Dashed lines indicate a translocation event.

mouse embryonic stem cells, leading to increases in the

expression levels of G1 cyclins and their CDK

part-ners, along with decreases in the levels of p21Cip1 and

p27Kip1 [52] Mitogenic responses through these

path-ways have been reported for a number of other Gq

-coupled receptors, including those for serotonin [53]

The proliferative actions of Gs-coupled GPCRs

mediated by these pathways have not been reported

Nevertheless, activation of the Gs-coupled

thyroid-stimulating hormone receptor (TSH) in thyrocytes

results in proliferation via an Akt-independent

path-way activated by the TSH receptor interaction with

PI3K, leading to the activation of p70S6K and mTOR

[54] A separate study has indicated that the TSH

receptor promotes PI3K pathway activation and DNA

synthesis by stimulating the association of PI3K with

Ras [55] Ras is known to bind to and activate several

PI3K subtypes [19], and itself is a major target of

GPCR activity [2]

In relation to GPCR control of proliferation, Akt

control of ERK has also been recorded For example,

agonist stimulation of the Gi-coupled adenosine A3

receptor expressed in human melanoma cells triggers

PI3K phosphorylation of Akt, leading to a reduction in

the levels of active, phosphorylated ERK1⁄ 2 and an

inhibition of cellular proliferation [56] ERKs are

known to regulate the transcriptional activity of several

transcription factors that control the expression of G1

cyclins and CDK inhibitors [2] A seemingly similar

PI3K⁄ ERK-dependent pathway is activated by SST2

receptors, leading to the induction of p27Kip1[57]

Akt mediation of GPCR-induced

survival and anti-apoptotic pathways

A key role of Akt is to facilitate cell survival and to

prevent apoptotic cell death In fact, dominant

nega-tive alleles of Akt reduce the ability of growth factors,

extracellular matrix and other stimuli to support cell

survival Conversely, the overexpression of Akt can

rescue cells from apoptosis [9] This is achieved by the

phosphorylation and inactivation of pro-apoptotic

factors such as Bad, caspase-9 and FH transcription

factors

Bad belongs to the Bcl2 family of apoptotic

pro-teins In some cell types, unphosphorylated Bad forms

a complex with pro-survival members of the Bcl2

family at the mitochondrial membrane, inducing the

release of cytochrome c from the mitochondria and

triggering caspase-mediated apoptosis Akt

phosphory-lation of Bad leads to its sequestration in the cytosol

by 14-3-3 proteins, preventing it from binding to its

partners at the mitochondrial membrane [9] Likewise,

Akt also phosphorylates and inactivates caspase-9, thereby inhibiting the terminal execution phase of apoptosis [12,58] In the absence of Akt activity, FH family members are found in the nucleus where they initiate apoptosis through the enhanced expression of specific pro-apoptotic Bcl2 family members Addition-ally, FH transcription factors promote the expression

of the tumour necrosis factor (TNF) receptor-associated death domain and of the TNF-related apoptosis-inducing ligand, leading to the activation of death-receptor signaling and caspase-mediated apopto-sis [59] Activated Akt phosphorylates FH family members, which are then exported from the nucleus and sequestered in the cytoplasm by their interaction with 14-3-3 proteins [12] Akt-dependent cell survival may also be achieved by the activation of the nuclear factor-jB (NF-jB) transcription factor and the direct phosphorylation and activation of the cAMP-response element binding protein These two transcription fac-tors have been implicated in the promotion of the expression of genes encoding survival proteins, such as c-myc, inhibitor-of-apoptosis proteins 1⁄ 2 and Bcl2 [9,60]

GPCR-mediated inhibition of apoptosis was observed many years ago when, for example, the acti-vation of muscarinic M3 receptors endogenously expressed in rat cerebellar granule neurons protected the cells against K+-induced apoptosis [61] In neuro-nal PC12 cells, agonist activation of exogenously expressed muscarinic M1 receptors protected against apoptosis induced by growth factor withdrawal [62] The intracellular pathways responsible for mediating these effects are gradually being revealed and it is now clear that Akt-dependent signaling is a vital avenue for the transmission of pro-survival, anti-apoptotic signals emanating from GPCRs For example, in transfected COS-7 cells both Gq-coupled M1 and Gi-coupled M2 muscarinic GPCRs are able to activate Akt and pre-vent UV-induced apoptosis [63], while the Go-coupled V2R pheromone receptor promotes the survival of vomeronasal stem cells via a pathway dependent on Akt and cAMP-response element binding protein acti-vation [64] Gs-coupled receptors have also been noted

to utilize Akt-dependent mechanisms to promote cell survival Adenosine acting through the A2A receptor transactivates the Trk neurotrophin RTKs, which in turn activate Akt and cell survival [65], while an uncharacterized Gs-coupled receptor for the peptide hormone ghrelin protects pancreatic b-cells against induced apoptosis via both Akt and MAPK pathways [66]

The GPCR-mediated signaling events downstream

of Akt have also begun to be characterized In

oligo-dendrocytes, carbachol (a nonselective muscarinic

receptor agonist) significantly reduces caspase-mediated

apoptosis by stimulating PI3K⁄ Akt pathways [67] The

role of caspases in GPCR-induced cell survival is

fur-ther confirmed by the ability of the peptide hormone

apelin to decrease the activation of caspase 9, as well

as caspases 3 and 8 In mouse osteoblasts, this

inhibi-tion of caspase activity and the apoptotic activity

induced by serum deprivation, steroids or TNF-a were

blocked by inhibitors of PI3K and Akt [68]

It is apparent that GPCRs also modify the

expres-sion and activity of members of the Bcl2 family of

pro-teins in order to regulate cell survival and apoptosis

As discussed above, the Akt-mediated phosphorylation

of FH transcription factors removes them from the

nucleus, preventing them from promoting the

expres-sion of pro-apoptotic Bcl2 proteins The initiation of

these pro-survival responses by GPCRs is little studied,

but there are indications that GPCR⁄ Akt ⁄ FH

tran-scription factor⁄ Bcl2 protein pathways are relevant to

cell survival For example, follicle stimulating hormone

is thought to play a role in follicular survival and

development in the ovary When expressed in

HEK-293 cells, the follicle stimulating hormone receptor

rap-idly promoted the phosphorylation and inactivation of

the FOXO1a FH transcription factor, probably by

Akt [69] The consequences of FOXO1a inactivation

were not examined in this study but other work has

described the ability of GPCRs to inhibit the

expres-sion of Bcl2 proteins In multiple cell types, the Gi

-coupled LPA receptors activate PI3K⁄ Akt ⁄ p70S6K

pathways as part of their cell survival mechanism [70]

It is suspected that the activation of these pathways by

LPA and sphingosine 1-phosphate receptors results in

the suppression of the cellular levels of the

pro-apopto-tic Bcl2 family member Bax [71] In PC12 cells, it was

determined that M4 receptors induced a Gbc

subunit-dependent activation of Akt and were able to augment

nerve growth factor (NGF)-mediated cell survival [40]

This Akt activation was accompanied by the

degrada-tion of TSC2 While not directly measured in this

study, removal of TSC2 would be expected to promote

mTOR activity mTOR is thought to affect apoptosis

and cell survival in several different ways, including by

regulating the expression levels of the anti-apoptotic

Bcl2 family member Bcl-XL[72]

It seems that given the correct conditions, GPCRs

can actually promote apoptosis In HeLa cells, this can

be achieved by an M1 receptor-mediated inhibition of

insulin receptor-stimulated Akt activation or by a

direct activation of caspase⁄ RhoA ⁄ Rho-associated

kinase pathways [73], possibly by up-regulating the

expression of the pro-apoptotic Bax [74], in contrast to

the previously noted suppression of the cellular levels

of Bax by the LPA and sphingosine 1-phosphate receptors [71] An alternative approach to the induc-tion of apoptosis has been adopted by the luteinizing hormone-releasing hormone, which inhibits the insulin growth factor-1 receptor-mediated activation of Akt Inhibition by the LHRH receptor in pituitary cells results in a reduction in Bad phosphorylation and a reduction in the ability of insulin growth factor-1 to rescue cells from apoptosis [75]

It is clear that Akt-dependent survival pathways rep-resent an attractive target for the development of anti-cancer agents In fact, inhibitors of mTOR not only cause cell cycle arrest but also promote apoptosis directly by sensitizing cells to the effects of DNA-dam-aging agents [72,76] The contribution that the regula-tion of GPCR activity may make to the modularegula-tion of these potentially therapeutic pathways is being inten-sively investigated [77,78] One approach to cancer therapy has been to target nonselectively the activity

of heterotrimeric G proteins using compound

BIM-46174 A variety of biochemical assays indicate that BIM-46174 inhibits the formation and⁄ or dissociation

of the Ga⁄ Gbc heterotrimeric complex Exposure of a variety of human cancer cells to BIM-46174 inhibits their growth by inducing caspase-dependent apoptosis

In mice, this drug seems to complement established chemotherapeutic regimes [79] Individual GPCRs have also been targeted For example, LPA receptors couple

to several different G protein subfamilies to activate Akt and the transcriptional activity of NF-jB In androgen-insensitive prostate cancer PC3 cells, this activation of Akt and NF-jB is required to escape cell death Therefore, it has been suggested that as NF-jB

is constitutively activated in prostate cancer, a strategy

of targeted disruption of the LPA⁄ Akt ⁄ NF-jB path-way may benefit androgen-insensitive prostate cancer treatment [80] In small cell lung cancer cells with con-stitutively active Akt signaling pathways, the applica-tion of the d-opioid receptor antagonist naltrindole promotes apoptosis This correlated with reduced levels of phosphorylation and activity of PI3K, Akt, glycogen synthase kinase 3b and FH transcription factors, as well as the up-regulation of several pro-apoptotic gene products [81]

Conclusions

There is little doubt that Akt is a crucial intermediary

in many intracellular signaling pathways initiated by diverse extracellular stimuli acting at several classes

of membrane-bound receptors Recent years have produced a growing body of evidence that clearly

establishes GPCRs as key initiators of the modulation

of Akt-dependent signaling events Furthermore, the

regulation of Akt-dependent cellular proliferation and

cellular survival in human cancer cells by numerous

GPCRs has opened up the possibility of controlling

cellular events through the use of ligands for a variety

of receptors The investigation of the applicability of

such an approach for therapeutic benefit is in its

infancy but, as we have described, progress has been

made with efforts to control growth and trigger

apop-tosis in human cancer cells by targeting heterotrimeric

G proteins and individual GPCRs [79,80] However,

experimental data obtained from transgenic and

knockout mice dictates that a cautious approach to

targeting Akt activity will be necessary Mice

defi-cient in Akt2 display insulin resistance and type-II

dia-betes-like syndrome, while both Akt1 and Akt2 are

required for platelet activation [8] Encouragingly,

expression of a dominant negative, kinase dead mutant

of Akt using an adenoviral vector selectively induced

apoptosis in tumor cells with elevated levels of Akt

activity but not in normal cells [82] This suggests that

unlike normal cells, tumor cells are dependent on

increased Akt activity for survival, indicating that

short-term inhibition of Akt signaling may not be toxic

to normal cells

As with many aspects of cellular signaling, much

remains to be uncovered in order to deepen our

understanding of how individual GPCRs functionally

interact with different G proteins to initiate a cascade

of events leading to the activation of different Akt

subtypes, which in turn trigger a multitude of

down-stream pathways Many of these GPCR-initiated

events are likely to be cell-type specific and

modu-lated by the actions of a host of other extracellular

and intracellular cues that must ultimately be

inte-grated to achieve the required biochemical⁄

physiologi-cal outcomes

Acknowledgements

This work was supported, in part, by grants from the

Research Grants Council of Hong Kong (HKUST

3⁄ 03C, HKUST 6443 ⁄ 06 m), the University Grants

Committee (AoE⁄ B-15 ⁄ 01), and the Hong Kong

Jockey Club YHW was a recipient of the Croucher

Senior Research Fellowship

References

1 Dummler B & Hemmings BA (2007) Physiological roles

of PKB⁄ Akt isoforms in development and disease

Biochem Soc Trans 35, 231–235

2 New DC & Wong YH (2007) Molecular mechanisms mediating the G protein-coupled regulation of cell cycle progression J Mol Signal 2, 2

3 Stambolic V & Woodgett JR (2006) Functional distinc-tions of protein kinase B⁄ Akt isoforms defined by their influence on cell migration Trends Cell Biol 16, 461– 466

4 Downward J (2004) PI 3-kinase, Akt and cell survival Semin Cell Dev Biol 15, 177–182

5 Hanada M, Feng J & Hemmings BA (2004) Structure, regulation and function of PKB⁄ AKT - a major thera-peutic target Biochim Biophys Acta 1697, 3–16

6 Okano J, Gaslightwala I, Birnbaum MJ, Rustgi AK & Nakagawa H (2000) Akt⁄ protein kinase B isoforms are differentially regulated by epidermal growth factor stim-ulation J Biol Chem 275, 30934–30942

7 Alessi DR, James SR, Downes CP, Holmes AB, Gaff-ney PR, Reese CB & Cohen P (1997) Characterization

of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Ba Curr Biol 7, 261–269

8 Fayard E, Tintignac LA, Baudry A & Hemmings BA (2005) Protein kinase B⁄ Akt at a glance J Cell Sci 118, 5675–5678

9 Nicholson KM & Anderson NG (2002) The protein kinase B⁄ Akt signalling pathway in human malignancy Cell Signal 14, 381–395

10 Chen YL, Law PY & Loh HH (2005) Inhibition of PI3K⁄ Akt signaling: an emerging paradigm for targeted cancer therapy Curr Med Chem Anti Canc Agents 5, 575–589

11 Liang J & Slingerland JM (2003) Multiple roles of the PI3K⁄ PKB (Akt) pathway in cell cycle progression Cell Cycle 2, 339–345

12 Vanhaesebroeck B & Alessi DR (2000) The PI3K-PDK1 connection: more than just a road to PKB Biochem J 346, 561–576

13 Zdychova J & Komers R (2005) Emerging role of Akt kinase⁄ protein kinase B signaling in pathophysiology

of diabetes and its complications Physiol Res 54, 1–16

14 Pierce KL, Premont RT & Lefkowitz RJ (2002) Seven-transmembrane receptors Nat Rev Mol Cell Biol 3, 639–650

15 Marchese A, George SR, Kolakowski LF Jr, Lynch KR

& O’Dowd BF (1999) Novel GPCRs and their endoge-nous ligands: expanding the boundaries of physiology and pharmacology Trends Pharmacol Sci 20, 370–375

16 Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sulton GG, Smith HO, Yandell M, Evans CA, Holt

RA et al (2001) The sequence of the human genome Science 291, 1304–1351

17 Drews J (2000) Drug discovery: a historical perspective Science 287, 1960–1964

18 Nambi P & Aiyar N (2003) G protein-coupled receptors

in drug discovery Assay Drug Dev Technol 1, 305–310

19 Schwindinger WF & Robishaw JD (2001)

Heterotrimer-ic G-protein bc-dimers in growth and differentiation

Oncogene 20, 1653–1660

20 Murga C, Fukuhara S & Gutkind JS (2000) A novel

role for phosphatidylinositol 3-kinase b in signaling

from G protein-coupled receptors to Akt J Biol Chem

275, 12069–12073

21 Wymann MP, Zvelebil M & Laffargue M (2003)

Phos-phoinositide 3-kinase signalling – which way to target?

Trends Pharmacol Sci 24, 366–376

22 Schafer B, Gschwind A & Ullrich A (2004) Multiple

G-protein-coupled receptor signals converge on the

epider-mal growth factor receptor to promote migration and

invasion Oncogene 23, 991–999

23 Ohtsu H, Dempsey PJ & Eguchi S (2006) ADAMs as

mediators of EGF receptor transactivation by G

pro-tein-coupled receptors Am J Physiol Cell Physiol 291,

C1–C10

24 Schafer B, Marg B, Gschwind A & Ullrich A (2004b)

Distinct ADAM metalloproteinases regulate G

protein-coupled receptor-induced cell proliferation and survival

J Biol Chem 279, 47929–47938

25 Shah BH, Neithardt A, Chu DB, Shah FB & Catt KJ

(2006) Role of EGF receptor transactivation in

phos-phoinositide 3-kinase-dependent activation of MAP

kinase by GPCRs J Cell Physiol 206, 47–57

26 Santiskulvong C, Sinnett-Smith J & Rozengurt E

(2001) EGF receptor function is required in late G1

for cell cycle progression induced by bombesin and

bradykinin Am J Physiol Cell Physiol 281, C886–

C898

27 Piiper A & Zeuzem S (2004) Receptor tyrosine kinases

are signaling intermediates of G protein-coupled

recep-tors Curr Pharm Des 10, 3539–3545

28 El Zein N, Badran BM & Sariban E (2007) The

neuro-peptide pituitary adenylate cyclase activating protein

stimulates human monocytes by transactivation of the

Trk⁄ NGF pathway Cell Signal 19, 152–162

29 Sumitomo M, Milowsky MI, Shen R, Navarro D, Dai

J, Asano T, Hayakawa M & Nanus DM (2001) Neutral

endopeptidase inhibits neuropeptide-mediated

transacti-vation of the insulin-like growth factor receptor-Akt cell

survival pathway Cancer Res 61, 3294–3298

30 Kumar RN, Ha JH, Radhakrishnan R & Dhanasekaran

DN (2006) Transactivation of platelet-derived growth

factor receptor a by the GTPase-deficient activated

mutant of Ga12 Mol Cell Biol 26, 50–62

31 Slomiany BL & Slomiany A (2005) Gastric mucin

secre-tion in response to b-adrenergic G protein-coupled

receptor activation is mediated by Src kinase-dependent

epidermal growth factor receptor transactivation

J Physiol Pharmacol 56, 247–258

32 Parsons SJ & Parsons JT (2004) Src family kinases, key

regulators of signal transduction Oncogene 23, 7906–

7909

33 Wang P & DeFea KA (2006) Protease-activated receptor-2 simultaneously directs b-arrestin-1-dependent inhibition and Gaq-dependent activation of

phosphatidylinositol 3-kinase Biochemistry 45, 9374–9385

34 Bajetto A, Barbero S, Bonavia R, Piccioli P, Pirani P, Florio T & Schettini G (2001) Stromal cell-derived factor-1a induces astrocyte proliferation through the activation of extracellular signal-regulated kinases

1⁄ 2 pathway J Neurochem 77, 1226–1236

35 Luttrell DK & Luttrell LM (2004) Not so strange bedfellows: G-protein-coupled receptors and Src family kinases Oncogene 23, 7969–7978

36 Bousquet C, Guillermet-Guibert J, Saint-Laurent N, Archer-Lahlou E, Lopez F, Fanjul M, Ferrand A, Fourmy D, Pichereaux C, Monsarrat B et al (2006) Direct binding of p85 to sst2 somatostatin receptor reveals a novel mechanism for inhibiting PI3K pathway EMBO J 25, 3943–3954

37 Beaulieu JM, Gainetdinov RR & Caron MG (2007) The Akt-GSK-3 signaling cascade in the actions of dopa-mine Trends Pharmacol Sci 28, 166–172

38 Imamura T, Huang J, Dalle S, Ugi S, Usui I, Luttrell

LM, Miller WE, Lefkowitz RJ & Olefsky JM (2001) b-Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport

J Biol Chem 276, 43663–43667

39 Revankar CM, Vines CM, Cimino DF & Prossnitz ER (2004) Arrestins block G protein-coupled receptor-medi-ated apoptosis J Biol Chem 279, 24578–24584

40 Wu EH, Tam BH & Wong YH (2006) Constitutively active a subunits of Gq ⁄ 11and G12 ⁄ 13families inhibit activation of the pro-survival Akt signaling cascade FEBS J 273, 2388–2398

41 Wu EH & Wong YH (2005) Involvement of Gi⁄ o pro-teins in nerve growth factor-stimulated phosphorylation and degradation of tuberin in PC-12 cells and cortical neurons Mol Pharmacol 67, 1195–1205

42 Moolenaar WH (1991) G protein-coupled receptors, phosphoinositide hydrolysis, and cell proliferation Cell Growth Differ 2, 359–364

43 Li S, Huang S & Peng SB (2005) Overexpression of G protein-coupled receptors in cancer cells: involvement in tumor progression Int J Oncol 27, 1329–1339

44 Schoneberg T, Schulz A, Biebermann H, Hermsdorf T, Rompler H & Sangkuhl K (2004) Mutant G protein-coupled receptors as a cause of human diseases Phar-macol Ther 104, 173–206

45 Sellers LA, Alderton F, Carruthers AM, Schindler M & Humphrey PP (2000) Receptor isoforms mediate oppos-ing proliferative effects through Gbc-activated p38 or Akt pathways Mol Cell Biol 20, 5974–5985

46 Goel R, Phillips-Mason PJ, Raben DM & Baldassare JJ (2002) a-Thrombin induces rapid and sustained Akt phosphorylation by b-arrestin1-dependent and