Protein kinase C-y (PKCy): it’s all

about location, location, location

Amnon Altman

Martin Villalba

Authors’ addresses

Amnon Altman1, Martin Villalba2,1Division of Cell Biology, La Jolla Institute

for Allergy and Immunology, San Diego,

CA, USA.2Institut de Genetique Moleculaire

de Montpellier, Montpellier,

France.

Correspondence to:

Dr Amnon Altman

Division of Cell Biology

La Jolla Institute for Allergy and Immunology

10355 Science Center Drive

San Diego, CA 92121

USA

Tel: þ1 858 558 3527

Fax: þ1 858 558 3526

e-mail: amnon@liai.org

Acknowledgements

We thank our many colleagues who contributed to our

work on PKCy over the years. The work reported herein

was supported by NIH grants CA35299, AI49888 and

CA95332, and by a grant from the Elizabeth Glaser

Pediatric AIDS Foundation. This is publication number

535 from the La Jolla Institute for Allergy and

Immunology.

Summary: Much progress has been made in understanding the functionof protein kinase C-y (PKCy) in the immune system since this Ca2þ-independent PKC isotype was isolated in 1993 as an enzyme that is highlyexpressed in T lymphocytes and in muscle cells. Biochemical and geneticapproaches revealed that, while dispensable for T-cell development, PKCyis required for the activation of mature T cells and for interleukin (IL)-2production. This deficiency results from impaired receptor-inducedstimulation of the transcription factors AP-1 and NF-kB. PKCy integratesT-cell receptor (TCR)/CD28 costimulatory signals, which are essential forproductive T-cell activation and, most likely, for prevention of T-cellanergy. A unique property of PKCy is its highly selective recruitment tothe central supramolecular activation complex (cSMAC) region of theimmunological synapse (IS) in antigen-stimulated T cells. Our workrevealed that this highly selective localization is not entirely dependenton phospholipase C (PLC) activity and diacylglycerol (DAG) production.Instead, a novel signaling pathway that requires functional Vav1, phos-phatidylinositol 3-kinase (PI3-K), the small GTPase Rac and actin cyto-skeleton reorganization regulates the localization and, perhaps, activationof PKCy. PKCy also provides a survival signal, which protects T cells fromapoptosis. Additional work is required to identify the immediate targetsof PKCy and its immune functions in vivo. This work is likely to validatePKCy as an attractive drug target.

Introduction: a bit of history

An important role for protein kinase C (PKC) in T-cell biology

has been suspected ever since that the following discoveries

were made:

� phorbol ester tumor promoters, for which PKC serves as

a major cellular receptor, synergize with Ca2þ ionophores

to induce T-cell activation and proliferation (1, 2); and;

� inositol phospholipid hydrolysis by phospholipase C (PLC),

which occurs early after T-cell receptor (TCR) triggering,

leads to production of diacylglycerol (DAG), a PKC-activat-

ing second messenger (3).

This notion was subsequently reinforced by findings that

depletion or pharmacological inhibition of PKC in T cells

Immunological Reviews 2003

Vol. 192: 53–63

Printed in Denmark. All rights reserved

Copyright � Blackwell Munksgaard 2003

Immunological Reviews0105-2896

53

reduces or abolishes TCR-induced T-cell activation and prolif-

eration (reviewed in 4). Coming on the heels of the isolation

of several genes encoding distinct PKC isotypes and the real-

ization that PKC enzymes constitute a growing family of

enzymes (reviewed in 5), we hypothesized that there must

exist another, yet to be discovered, PKC isotype, with a unique

and selective role in T-cell biology. In 1989, we began a search

for this putative PKC isotype and, using the polymerase chain

reaction (PCR) method in conjunction with degenerate pri-

mers corresponding to conserved sequences within the cataly-

tic domain, we fairly quickly isolated a partial cDNA sequence

from T cells that was homologous to but distinct from other

known PKCs. We tentatively named this new isotype PKCy.

Subsequent efforts by Dr Gottfried Baier, a postdoctoral

research fellow who joined our laboratory in February 1990,

led to cloning of the human PKCy full-length cDNA and its

initial functional characterization (6), revealing that PKCy is a

member of the Ca2þ-independent, novel PKC (nPKC) subfam-

ily. Others have independently isolated the cDNAs encoding

mouse and human PKCy at about the same time (7, 8). Work

carried out since then in our laboratory and by others has

revealed that PKCy plays an important and nonredundant

role in the activation of mature T cells. This work has recently

been reviewed in a number of publications (9–11). Here, we

will focus our attention on the unique properties of PKCy in

T cells which distinguish it from other T-cell-expressed PKC

enzymes, and discuss our recent work on how these properties

reflect a specialized, and perhaps novel, mechanism that reg-

ulates the intracellular localization and, hence, the proper

function of this enzyme.

Functions of PKCy

Soon after isolating human PKCy, we found that it is expressed

in a relatively selective manner in T cells and in muscle

cells (6). This expression profile is unique and different from

all other PKC isotypes. We then began a search for specialized

functions of this enzyme in T cells. One potential target was

the transcription factor activator protein-1 (AP-1), which plays

an important role in the induction of many immune-related

genes, including the interleukin-2 (IL-2) gene, and is known

to be activated by phorbol esters (12). We found that PKCy,

but not other PKC isotypes that were tested, activated AP-1 and

that a dominant negative (kinase-inactive) PKCy mutant

blocked phorbol ester-stimulated AP-1 activity (13). Consis-

tent with this finding, several groups later reported that c-Jun

N-terminal kinase (JNK), which contributes to AP-1 activation

by phosphorylating c-Jun, is also an activation target of PKCy

but not other PKC isotypes (14–16). However, the physiolo-

gical relevance of PKCy-mediated JNK activation is unclear,

since peripheral T cells from PKCy-deficient mice display

intact TCR/CD28-induced JNK activation in the face of defec-

tive AP-1 activation (17).

Transfection of Jurkat T cells with a combination of consti-

tutively active PKCy and calcineurin plasmids was found to

induce synergistic activation of the IL-2 gene and, conversely,

dominant negative PKCy selectively inhibited activation of the

IL-2 promoter (15, 16). The requirement for PKCy was spe-

cific, since no other PKC reproduced these effects. This activity

of PKCy was mainly attributed to nuclear factor of activated

T cells (NFAT) activation, consistent with the fact that the

proximal NFAT site in the IL-2 promoter cooperatively binds

a complex of AP-1 plus NFAT (18).

More recent studies utilizing transfection approaches

revealed that PKCy also plays an important and selective role

in activating the transcription factor NF-kB in T cells (19, 20).

Similarly, PKCy stimulates and is required for TCR/CD28-

induced activation of the CD28 response element (RE) in the

IL-2 gene promoter (20), a site that binds a combination of

NF-kB and AP-1 (21, 22). Functional analysis of peripheral

T cells from PKCy-deficient mice confirmed the important

roles of PKCy revealed by transfection studies (17). Thus,

PKCy–/– mature T cells displayed a severe defect in TCR/

CD28-induced IL-2 production and proliferation. This defect

was attributed to deficient stimulation of both AP-1 and NF-kB.

Surprisingly, however, T-cell development (both negative and

positive selection) was intact in these mice, suggesting that

another PKC or perhaps a PKC-independent pathway is

required for T-cell development.

PKCy, the immunological synapse and lipid rafts

T-cell activation requires sustained TCR interaction with MHC-

bound peptide antigen at the T cell–antigen-presenting cell

(APC) contact region. Productive interaction results in bio-

chemical changes and reorganization of specific membrane

domains, which leads to the formation of a highly ordered

signaling complex at the contact site, the so-called immuno-

logical synapse (IS) (23). Formation of a functional IS also

involves the assembly of signaling complexes consisting of

TCRs, costimulatory accessory receptors (such as CD28, CD4/

CD8 or leukocyte function-associated antigen-1 (LFA-1)),

and intracellular signaling effector proteins (23–25), reor-

ganization of the actin cytoskeleton (26), and clustering of

specialized membrane microdomains or lipid rafts (27, 28).

A more detailed analysis of the T cell–APC contact region

Altman & Villalba � Protein kinase C-y (PKCy) location

54 Immunological Reviews 192/2003

revealed compartmentalization of molecules in at least two

distinct identifiable areas of the synapse, the so-called central

supramolecular activation complex (cSMAC) and peripheral

SMAC (pSMAC) (24). While the cSMAC is characterized by

clustering of TCR and major histocompatibility complex

(MHC) molecules on the T cell and APC surfaces, respectively,

the pSMAC in these two cell types is enriched with LFA-1

integrins and their intercellular adhesion molecule (ICAM)-1

counter-receptors, respectively. The spatial organization and

stability (or duration) of the IS determine the functional out-

come of TCR engagement and underlie the fundamental phe-

nomenon of differential T-cell signaling (29).

A major contribution to our understanding of the role of

PKCy in T-cell biology was made when it was discovered that

engagement of antigen-specific T cells by peptide-presenting

APCs led to a rapid, stable and high-stoichiometry localization

of PKCy but not other T-cell-expressed PKCs (bI, d, e, Zand z) to the T cell–APC contact site (30) and, more specifically,

to the cSMAC (24). This localization occurs at a high stoichio-

metry and lasts for at least 2–4 h, suggesting that it could play

an important role in propagating and extending activation

signals that are required for T-cell commitment to IL-2 pro-

duction, long after early tyrosine phosphorylation events have

been extinguished. The cSMAC clustering of PKCy correlated

with its catalytic activation, and only occurred upon produc-

tive activation of T cells, i.e. upon exposure to APCs that were

fed with optimal antigen concentrations leading to efficient

proliferation. In contrast, altered peptide ligands or low pep-

tide concentrations that induced weak or no detectable pro-

liferation did not promote PKCy recruitment to the cSMAC

(30). Coclustering of talin and tubulin, and formation and

reorientation of the microtubule-organizing center (MTOC)

were also observed under these conditions. Subsequently, it

became clear that signaling molecules on the inner side of the

cell membrane also segregate into two nonoverlapping regions

characterized by PKCy and Lck at the cSMAC, just below the

TCR, and talin molecules in the peripheral zone, where they

can directly interact with the LFA-1 cytoplasmic tail (23, 24).

Another prominent feature of PKCy is its activation-induced

translocation into lipid rafts (31). The structural basis for this

association is unclear, but our work revealed that this translo-

cation is enhanced by CD28 costimulation and that it requires

functional Lck but not ZAP-70. Similar to PKCy itself, these

lipid rafts also cluster at the IS in antigen-stimulated T cells

but, unlike PKCy, the rafts are not restricted to the cSMAC.

Thus, lipid raft translocation of PKCy per se is unlikely to

account entirely for the very specific concentration of the

enzyme in the cSMAC during antigen stimulation. Neverthe-

less, receptor-induced clustering of lipid raft may serve as an

important driving force that promotes the initial translocation of

PKCy to the IS, where additional mechanism(s) may function to

selectively recruit into a specific subregion of the IS, i.e. the

cSMAC. This notion is supported by our finding that disruption

of cholesterol-rich lipid rafts by pretreatment with relatively low

concentrations of b-methyl cyclodextrin (MCD), which still

allows some conjugate formation between antigen-specific

T cells and APCs, prevents the translocation of PKCy to the IS

(31). A higher MCD concentration abrogates the formation of

stable T cell–APC conjugates, but this effect can be partially

reversed by increasing the immunogenic peptide concentration

(i.e. increasing TCR occupancy), suggesting that raft clustering at

the IS may be especially important in promoting the proper

cSMAC localization of PKCy and, perhaps, the organization of a

mature IS under physiological conditions of low TCR occupancy.

The importance of PKCy membrane microdomain

localization for its function

The unique functions of distinct PKC isotypes are determined

by their substrate specificity and their intracellular localization.

Distinct localization patterns dictate the accessibility of each

PKC isotype to its regulators and substrates, including PKC

association with potential receptors for activated C-kinase

(RACK) proteins (32, 33). In general, relatively little is

known about the precise mechanisms that regulate these

events and, hence, the individual functions of each PKC iso-

type. However, once the important function of PKCy was

clearly established, considerable effort was and continues to

be dedicated to understanding the relationship between the

changes in PKCy localization that result from TCR/costimula-

tory receptor ligation and its proper function.

Our work revealed that the membrane and lipid raft trans-

location of PKCy may be important for its proper function,

since the isolated catalytic domain of PKCy, which is fully

active in vitro, failed to translocate into lipid rafts and activate

NF-kB (31). This defect was rescued when an Lck-derived

raft-targeting signal was fused to the catalytic region. Other

studies suggest that PKCy actually needs to reside within the

cSMAC in order to function properly, and that CD28 costimu-

lation plays a unique and nonredundant role in effecting this

highly selective localization (34: Sedwick C, Miller J. Personal

communication). These studies demonstrated that CD28 cost-

imulation led to a defined concentration of PKCy in the

cSMAC, whereas LFA-1 costimulation resulted in a diffuse

pattern of PKCy and LFA-1 localization, which is characteristic

of an immature synapse. More importantly, only the focused

Altman & Villalba � Protein kinase C-y (PKCy) location

Immunological Reviews 192/2003 55

concentration of PKCy in the cSMAC correlated with NF-kB

activation as revealed by confocal analysis of the nuclear trans-

location of this transcription factor (Sedwick C, Miller J.

Personal communication). These results are consistent with our

earlier findings that CD28 provides unique and essential sig-

nals required for the proper localization and activation of PKCy(19, 31, 35). The biochemical nature of these signals is

unclear, but one likely candidate is phosphatidylinositol 3-

kinase (PI3K), which inducibly associates with the cytoplasmic

tail of CD28 and becomes activated by crosslinking this costi-

mulatory receptor (36). Indeed, our work has demonstrated

that inhibition of cellular PI3K activity impairs the CD3/CD28-

induced membrane translocation of PKCy (see below).

What’s so special about PKCy?

Among all T-cell-expressed PKC isotypes, only PKCy trans-

locates to the IS (cSMAC) upon peptide/MHC stimulation, sug-

gesting that a unique mechanism exists to regulate this highly

selective localization and, perhaps, downstream functions of

PKCy. Normally, membrane translocation and subsequent acti-

vation of conventional, Ca2þ-dependent PKC enzymes (cPKC)

and novel, Ca2þ-independent isotypes (nPKC) requires their

conserved C1 domain, which binds the second messenger

DAG formed in the inner leaflet of the plasma membrane as

a result of PLC activation by various receptors. The importance

of this event was demonstrated by findings that mutations in

the C1 domain of several members of the PKC family, e.g.

PKCa (a cPKC) and PKCd (an nPKC), abolish DAG/phorbol

myristate acetate (PMA) binding in vitro and/or PMA-mediated

membrane translocation (37, 38). While DAG-mediated mem-

brane recruitment could play a role in the translocation and

activation of PKCy as well, it is difficult to explain how

DAG binding alone, which is relatively nonselective, could

account for the highly specific recruitment of PKCy to the

cSMAC in the IS. This high degree of selectivity implicates an

additional undefined mechanism that either cooperates with

PLC-generated DAG, or acts exclusively, to recruit PKCy to,

and activate it in, specific membrane microdomains, i.e. the

cSMAC (24, 30) or lipid rafts (31, 39). In recent years we have

dedicated considerable efforts to elucidating the regulatory

mechanism responsible for this highly selective localization

of PKCy triggered by TCR/CD28 costimulation.

The PKCy-cytoskeleton connection

Most T-cell-expressed PKC isotypes, including PKCy, undergo

at least a transient plasma membrane translocation in response

to TCR ligation (or phorbol ester treatment). However, unlike

other PKCs, only PKCy translocates to the detergent-insoluble

cellular fraction, which mostly represents the actin cytoskele-

ton (35, 40). This unique behavior of PKCy led us to examine

the potential role of the major hematopoietic cell-specific Rac/

Cdc42 guanine nucleotide exchange factor (GEF), Vav1 (41),

in regulating the localization and/or activation of PKCy. This

possibility was consistent with the demonstrated essential role

of Vav1 in inducing TCR capping (42, 43). Antibody-mediated

crosslinking of the TCR leads to aggregation of receptor

complexes and their clustering at a distinct region of the cell

membrane in a so-called cap structure. TCR capping is an active

process mediated by TCR-induced reorganization of the actin

cytoskeleton, and this process is thought to be analogous, in

at least some respects, to TCR translocation to the IS during

antigen stimulation (26).

Using transfected Jurkat T cells, we found that a dominant

negative PKCy mutant blocked a number of growth signals

that are normally induced by overexpression of Vav1, namely,

activation of JNK, the IL-2 gene promoter and NFAT or AP-1

reporter genes (35). Nevertheless, the same mutant did not

inhibit the basal or anti-CD3-induced actin polymerization

induced by Vav1 overexpression. Conversely, a dominant

negative Vav1 mutant did not significantly inhibit the same

signaling events induced by a constitutively active PKCymutant, tentatively placing PKCy downstream of Vav1 in

these growth signaling pathways, but not in the pathway

leading from Vav1 to actin cytoskeleton reorganization. Domi-

nant negative PKCy (but not dominant negative PKCa, eor z mutants) also inhibited Vav1-induced up-regulation of

CD69 expression. Additional experiments revealed that Vav1

promoted translocation of PKCy from the cytosol to the

membrane and cytoskeleton, and that the anti-CD3-stimulated

PKCy translocation was inhibited by dominant negative Vav1

or Rac1 mutants, as well as by cytochalasin pretreatment (35).

Finally, overexpression of Vav1 or a constitutively active

Rac1 mutant stimulated the catalytic activity of PKCy. We

later confirmed these findings generated in Jurkat T cells by

demonstrating that combined anti-CD3/CD28 stimulation,

which induced prominent PKCy translocation to a membrane

cap-like structure in wild type T cells, failed to induce such

a response in Vav1–/– T cells (39).

Based on these results, we concluded that Vav1 and PKCyfunction in overlapping but not identical pathways. We

proposed that Vav1, when activated by TCR-coupled protein

tyrosine kinases (PTKs), activates Rac and/or Cdc42, leading

to actin polymerization and TCR capping, a process that is in

itself PKCy-independent. These Vav1-mediated events are

Altman & Villalba � Protein kinase C-y (PKCy) location

56 Immunological Reviews 192/2003

essential for the translocation of PKCy and its colocalization

with the TCR in the SMAC, as well as for its enzymatic

activation. Direct or indirect association of PKCy with a cytos-

keletal protein, which is consistent with our findings, could

represent one mechanism through which Vav may promote

PKCy translocation and activation. According to this model,

the dependence of downstream signaling events, such as JNK

activation and IL-2 gene induction on Vav1 and Rac, reflects

the essential role of these proteins in inducing PKCy mem-

brane localization and activation via their well-established

effects on the actin cytoskeleton. The novel aspects of this

study (35) were the functional linkage of PKCy to Vav1 and

Rac in a T-cell growth regulatory pathway and the apparent

dependence of Vav1 on intact PKCy function in this pathway.

This link between Vav1 and PKCy provides a mechanistic

basis for the functional and/or physical interaction between

these two proteins, which was documented in other studies

(44, 45).

The role of PLCg1 activation and DAG production

vs. the role of PI3K

The discovery of a unique Vav/Rac-dependent pathway

required for the recruitment of PKCy to the membrane (or

IS?) and perhaps its activation, as discussed above (35), raised

the question whether this pathway represents an alternative

PKCy activation mechanism that is independent of PLCg1

activation and DAG production, the latter representing the

conventional and well-established pathway for the membrane

translocation and activation of other nPKCs and cPKCs. We

recently addressed this question by using three independent

approaches for inhibiting the function of PLC in normal or

Jurkat T cells: a selective PLC inhibitor (U73122), a PLCg1-

deficient Jurkat T-cell line, or a dominant negative PLCg1

mutant (46). We demonstrated that CD3/CD28-induced mem-

brane recruitment and activation loop phosphorylation of PKCyare partially independent of PLC, since these responses remained

largely intact when PLC activity was inhibited. In contrast, PLC

inhibition blocked the membrane translocation of a represen-

tative cPKC, PKCa. As an additional control for the efficiency

of PLC inhibition, we demonstrated that the dominant-negative

PLCg1 mutant blocked anti-CD3- or Vav1-induced NFAT

activation, while failing to inhibit the membrane transloca-

tion of PKCy or actin capping observed by confocal micro-

scopy (46).

The finding that, in contrast to PKCa, the membrane

translocation and phosphorylation of PKCy is not entirely

dependent on PLC activity led us to address the possibility

that some functional manifestations of PKCy may occur

‘upstream’ of PLC activation and, moreover, that PKCy may

regulate PLC function via some positive feedback regulatory

loop. Early experiments supported this potentially novel func-

tion by demonstrating that a constitutively active PKCy mutant

induced membrane and some cytoskeleton translocation of

PKCa, a known PLC/DAG-dependent event. Similarly, we

found the TCR/CD28-induced membrane translocation of

PKCa to be partially but consistently deficient in PKCy-

deficient T cells (A. Altman et al., submitted for publication).

Upon extending these studies, we were surprised to find that

stimulus-induced PLCg1 tyrosine phosphorylation and Ca2þ

mobilization were significantly decreased in the same cells,

indicating that PKCy is required for optimal activation or

maintenance of Ca2þ signaling pathways in primary T cells.

In further support of this regulatory pathway, we found that

a dominant negative PLCg1 mutant blocked activation of an

AP-1 reporter gene induced by PKCy, but not by PKCa,

thereby potentially placing PLCg1 downstream of PKCy but (as

expected) upstream of PKCa. To further elucidate the mechan-

ism of this apparent PKCy-mediated regulation of PLCg1, we

investigated the role of Tec-family kinases, which are known

to positively regulate the function of PLCg1 and Ca2þ signaling

pathways in T cells (47). We found that transfected wild type

Tec, but not Itk or Rlk, potently activated AP-1, a known

physiological target of PKCy (but not NF-kB) and that, con-

versely, a dominant negative Tec mutant suppressed PKCy (but

not PKCa)-induced AP-1 activation. We also obtained preli-

minary evidence of a physical interaction between PKCy and

Tec in cotransfected 293T cells. Additional molecular details of

this novel regulatory function by PKCy are currently under

study, but our results demonstrate that there potentially exists

in T cells a PKC cascade, in which PKCy functions upstream

of PLCg1 to positively regulate the activity of the latter and

Ca2þ signaling via Tec, thereby contributing to the activation

of PKCa and AP-1.

Since activation of PI3-K has been linked to both Vav1 and

Rac in T cells, we also addressed the role of PI3K in the

receptor-induced membrane recruitment of PKCy, using a

selective pharmacological inhibitor of PI3K, LY294002. This

compound inhibited the anti-CD3/CD28-membrane trans-

location of PKCy, but not PKCa, in peripheral blood T cells

(46). One potential PI3K target that could play a role

in the membrane recruitment of PKCy is PI3K-dependent

kinase-1 (PDK1), which associates with, and is responsible for

activation loop phosphorylation of, different PKC enzymes

(48–50). PDK1 and PKC need to be corecruited to the membrane

through interaction with their respective membrane-localized

Altman & Villalba � Protein kinase C-y (PKCy) location

Immunological Reviews 192/2003 57

allosteric activators in order for this phosphorylation to be

efficient (48, 51–53). Therefore, a potential scenario could

be envisaged in which PKCy associates with PDK1 and then is

recruited to the membrane via the pleckstrin-homology (PH)

domain of the latter. Consequently, we examined the localiza-

tion of PDK1 and PKCy in unstimulated or TCR-stimulated

T cells. We found that anti-CD3/CD28 stimulation, which

induced marked membrane translocation of PKCy, failed to

cause similar detectable translocation of PDK1, suggesting that

PDK1 does not contribute significantly to the plasma mem-

brane recruitment of PKCy (46). Consistent with this notion,

we also found that coexpression of PDK1 with PKCy did not

enhance the PKCy-induced activation of NF-kB and AP-1

reporter genes. Nevertheless, we found that PDK1 overexpres-

sion enhanced the membrane and cytoskeleton translocation

of PKCy, but this effect was only partially sensitive to a PI3K

inhibitor. Together, these results suggested that, although

PDK1 may be involved in the maturation (perhaps via activa-

tion loop phosphorylation) of PKCy in a similar manner to

other PKC enzymes, it does not directly translocate PKCy to the

membrane by associating with it in T cells.

Regulation of PKCy by tyrosine phosphorylation

Triggering of the TCR was found to lead to rapid phos-

phorylation of PKCy on tyrosine, predominantly on Tyr90 in

the regulatory domain (54). Phosphorylation was mediated

by Lck, which also interacted directly with the PKCy regulatory

domain as demonstrated by pull-down with GST fusion

proteins, coimmunoprecipitation and an overlay assay.

We observed basal Lck association with PKCy in resting

cells, but this association increased following T-cell activation

and involved both the SH2 and SH3 domains of Lck. The

functional relevance of this tyrosine phosphorylation was

addressed by mutating the relevant tyrosine residue into

phenylalanine and testing the effects of this mutation on

PKCy-dependent functions. We found that overexpressed

constitutively active PKCy (PKCy-A148E) increased the

proliferation rate of Jurkat cells and synergized with iono-

mycin in induction of NFAT activity. In contrast, a Tyr90-

to-Phe mutation markedly reduced both activities (54), but

it did not reduce the ability of the same mutant to activate an

AP-1 reporter gene. These results suggest that the physical

association of Lck with PKCy and the Lck-induced tyrosine

phosphorylation of PKCy represent physiologically relevant

events that regulate PKCy during TCR-induced T-cell

activation.

How does PKCy activate AP-1 vs. NF-kB?

As noted earlier, activation of both NF-kB and AP-1 is deficient

in PKCy–/– T cells (17), indicating that PKCy plays a physio-

logically relevant, nonredundant role in the activation of these

two transcription factors, which, in turn, is essential for induc-

tion of the IL-2 and other cytokine genes in activated T cells.

Thus, considerable effort is currently being made to identify

the direct targets and intermediates in the signaling pathways

leading from PKCy to activation of AP-1 and NF-kB.

With regard to NF-kB, we and others reported that PKCyactivates inhibitor of kB (IkB) kinase-b (IKKb), but not IKKa,

and that a dominant negative IKKb was more potent than a

dominant negative IKKa mutant in inhibiting the activation of

NF-kB or CD28 RE reporter genes induced by CD3/CD28

costimulation or by a constitutively active PKCy mutant (19,

20). Another signaling event in the NF-kB pathway, which

occurs downstream of IKK activation, i.e. the CD3/CD28-

induced nuclear translocation of cytosolic NF-kB proteins,

was also deficient in PKCy–/– T cells, and it was reduced by

pretreating T cells in vitro with rottlerin, a relatively selective

inhibitor of PKCy (and possibly other nPKCs), but not by an

inhibitor of cPKCs, Go6976 (19). Degradation of IkB, which

follows the IKK-mediated phosphorylation of this protein, was

similarly reduced in stimulated PKCy–/– T cells (17).

Although coexpression of PKCy induces phosphorylation and

activation of IKKb in transfected cells, it is very difficult, if not

impossible, to demonstrate direct phosphorylation of IKKb by

purified PKCy in vitro, suggesting that IKKb is not a direct

substrate of PKCy. Instead, there must be some other immediate

substrate of PKCy which is placed upstream of IKK and leads to

its activation. The nature of this potential PKCy target is

unknown, but one candidate is the Akt (PKB) kinase, which

activates NF-kB and CD28RE/AP-1 in T cells (55). However,

the effects of PKCy and Akt differ from each other. First, while

constitutively active PKCy activated NF-kB and CD28RE/AP-1

in unstimulated cells, activation mediated by wild type Akt or

even by active, membrane-targeted Akt required stimulation

with anti-TCR antibody plus phorbol ester. Second, unlike

PKCy, Akt did not activate NFAT or AP-1. Finally, Akt, but not

PKCy, was capable of activating IKKa (55). These differences

are more consistent with the notion that Akt is not a target of

PKCy. In fact, more recent work indicates that PKCy and Akt

physically interact with each other and functionally cooperate to

activate NF-kB in T cells. However, these two kinases do not

phosphorylate each other (56, 57).

Another possibility is that the target of PKCy in the NF-kB

pathway is some mitogen-activated protein kinase kinase

Altman & Villalba � Protein kinase C-y (PKCy) location

58 Immunological Reviews 192/2003

kinase (MAP3K). Potential candidates include several MAP3Ks

that have been implicated in NF-kB activation in T cells:

MEKK1, NIK and Cot (58, 59). NIK has tentatively been placed

downstream of Cot in the TCR/CD28 signaling pathway leading

to NF-kB activation (58). The findings that PKCy selectively

activates both the JNK/AP-1 pathway (13–16, 35) and the IKK/

IkB/NF-kB pathway (17, 19, 20), and that MEKK1 mediates

cross-talk between the JNK and IKK cascades (59), raises the

possibility that MEKK1 is a target for PKCy in T cells. However,

so far we have not been able to block the PKCy-mediated

activation of CD28RE/AP-1 in T cells by coexpressing a dominant

negative MEKK1 mutant (Y. Li, unpublished data). In contrast,

dominant negative Cot was capable of inhibiting this activation.

We also found that recombinant kinase-inactive Cot is phosphor-

ylated by PKCy in vitro (Y. Li et al., manuscript in preparation).

This finding raises the possibility that Cot is a PKCy target

in T cells, and it is consistent with the ability of Cot to

up-regulate expression of the IL-2 gene and to simultaneously

activate several kinases and transcription factors (58, 60–64)

that are also induced by PKCy, i.e. JNK, ERK, NFAT, and

NF-kB. However, even if Cot plays a role in PKCy-mediated

NF-kB activation, this role appears to be redundant, since

T cells of Cot–/– mice are phenotypically and functionally

normal (65).

Recently there has been interest in two types of PDZ

domain-containing proteins that may couple PKCy to NF-kB

activation in T cells. The first is Bcl10, which is an NF-kB-

activating adapter protein. T and B cells from Bcl10-deficient

mice display a defect in NF-kB activation (66). Another pro-

tein is CARD11/CARMA1, a membrane-associated guanylate

kinase (MAGUK) family member, which also contains a cas-

pase recruitment domain (CARD) and an SH3 domain (67,

68). Several groups demonstrated very recently that CARD11

plays an essential role in transmitting TCR/CD28 signals lead-

ing to NF-kB activation and IL-2 production (69–71).

Furthermore, like PKCy, CARD11 also translocated to lipid

rafts in stimulated T cells and was associated with Bcl10 and

the TCR complex (69). A potential link between CARD11 and

PKCy is also implicated by the finding that coexpression of a

constitutively active PKCy mutant together with CARD11 (or

Bcl10) synergistically activated NF-kB in T cells (71). How-

ever, it remains to be determined whether PKCy forms a

complex with CARD11 and/or Bcl10 in T cells. If such a

complex is indeed induced in TCR/CD28-stimulated T cells,

it could have an important role in transducing PKCy signals to

downstream targets in the NF-kB pathway. CARD11 recruits

Bcl10 to the plasma membrane (71) and localizes itself to lipid

rafts upon stimulation (69). Furthermore, as a member of the

MAGUK protein family, CARD11 may be ideally positioned

to link intracellular signaling molecules to the cytoskeleton

and to plasma membrane receptors, a well-known property

of other MAGUK proteins (72–74). It is also possible that

the CARD11/Bcl10 complex may couple PKCy to the IKK

signalsome, since this complex was proposed to bind the

IKK complex (67, 71). Consistent with such a model, it

was found that PKCy associates with the IKK complex,

which also localizes to membrane lipid rafts in stimulated

T cells (75).

Available evidence indicates that bifurcation of the signaling

pathways leading to NF-kB vs. AP-1 occurs at some undefined

point downstream of PKCy. Thus, CARD11 deficiency impairs

NF-kB, but not AP-1 activation (70, 71). Conversely, we find

that Tec kinase acts downstream of PKCy to activate AP-1 but

not NF-kB (see above). However, very little is known about

the mechanism that links PKCy to AP-1 activation. One obvious

link is JNK, which can be activated by PKCy (14–16, 35),

since JNK is known to phsophorylate c-Jun on two serine

residues in its transactivating domain, thereby contributing

to AP-1 activation (76, 77). However, the finding that recep-

tor-stimulated JNK activation is intact in PKCy–/– T cells,

despite the deficient AP-1 activation in the same cells (17),

suggests that an alternative, JNK-independent pathway couples

PKCy to AP-1 activation. One interesting candidate that may

link PKCy to AP-1 activation is the MEF2 transcription factor

family. Members of the MEF2 family promote AP-1 by bind-

ing to the c-Jun promoter and up-regulating its expression

(78, 79), including in T cells (81). T cells are known to

abundantly express MEF2D (80, 81) and, consistent with

this notion, we recently found that a constitutively active

PKCy mutant but not similar mutants of other PKC isotypes

(a, e, z), potently activates a MEF2 reporter gene (Y. Li et al.,

unpublished data).

In a recent yeast two-hybrid screen for PKCy-interacting

proteins, we isolated an STE20-related novel MAP3K, which

appears to function selectively in PKCy-mediated activation of

AP-1 (Y. Li et al., manuscript in preparation). This kinase

synergizes with limiting amounts of constitutively active

PKCy to activate AP-1 but not NF-kB. Conversely, an inactive

mutant of this kinase inhibited in a dose-dependent manner

the PKCy-induced activation of AP-1 but not NF-kB.

Furthermore, CD3/CD28-induced activation of this kinase is

deficient in PKCy–/– T cells, and the recombinant kinase

is directly phosphorylated in vitro by purified PKCy. We have

mapped the tentative phosphorylation site of this kinase,

and experiments are in progress to further evaluate its functional

significance in T cells.

Altman & Villalba � Protein kinase C-y (PKCy) location

Immunological Reviews 192/2003 59

A role for PKCy in T-cell survival?

Many studies have indirectly implicated a role for members

of the PKC family in protection against activation-induced

cell death (AICD). For example, PKC activation by phorbol

ester treatment protects various cell types from apoptosis

(82–85). T cells are sensitized to Fas-mediated apoptosis by

the PKC inhibitor, bisindolylmaleimide VIII (86). However,

until recently, the identity of the relevant PKC isotype(s) has

not been known. Given that CD28 costimulation can provide

a survival signal that protects T cells from AICD (87) and also

appears to have an important role in productive activation of

PKCy (19), we examined whether PKCy provides a T-cell

survival signal. We and another group recently demonstrated

that PKCy can promote T-cell survival, predominantly by

phosphorylation and inactivation of BAD (88, 89), thereby

protecting the cells from Fas-induced apoptosis. Another

nPKC, PKCe, was less efficient than PKCy, whereas PKCa and

PKC� were relatively ineffective. Rottlerin, which inhibits

PKCy in a relatively selective manner (and probably other

nPKCs, of which PKCd is barely expressed in T cells), syner-

gized with low concentrations of anti-Fas antibodies to induce

rapid and marked apoptosis of Jurkat T cells, a mouse T-cell

hybridoma, or activated human peripheral blood T cells (89).

In contrast, an inhibitor of cPKCs (Go6976) did not show this

synergistic activity. Dominant negative PKCy as well as

pharmacological inhibitors of PKC abolished the protective

effect of phorbol ester and promoted Fas-mediated apoptosis.

Both PMA and overexpressed constitutively active PKCy or

PKCe induced BAD phosphorylation at Ser112. Additional stud-

ies by Villalba et al. demonstrated that engagement of Fas

by specific antibodies led to a transient activation of PKCy,

which was later followed by caspase-3-dependent cleavage of the

enzyme (89). In addition, Fas ligation resulted in proteasome-

mediated degradation of PKCy and inactivation of its catalytic

fragment, events that preceded the onset of cell apoptosis.

Leukemic Jurkat T cells often show a higher basal PKCytranslocation to the membrane by comparison with normal,

nonleukemic T cells, in which PKCy is localized exclusively in

the cytoplasm (90). This translocation is normally associated

with activation of the enzyme. Since many T-cell leukemias

display markers of an activated phenotype, e.g. the IL-2 recep-

tor, it is conceivable that PKCy may be constitutively active in

some leukemic cells. Therefore, identification of selective

PKCy inhibitors could lead to development of drugs that may

enhance apoptosis and elimination of malignant T cells by

inhibiting the function of PKCy. This increased apoptosis

may lead to two additional and interrelated beneficial effects.

First, reduction of the tumor mass may facilitate establishment

of T-cell immunity against tumor-specific antigens. Second,

APCs that phagocytose apoptotic tumor cells are potent tumor

vaccines (91). Although all T-cell leukemia lines examined to

date express PKCy (6), its activity status and localization in

these cells remain to be analyzed.

Another potential role of PKCy in the survival of leukemic

T cells is related to the acquisition of a multidrug resistance

(MDR) phenotype. Cells with an MDR phenotype are charac-

terized by cross-resistance to a broad spectrum of structurally

and functionally unrelated drugs. This phenotype probably

arises through overexpression of P-glycoprotein (P-gp), an

ATP-dependent transmembrane drug transporter, which

reduces intracellular drug concentrations by pumping sub-

strates out of the cells (92). PKCy expression is increased in

cells with an MDR phenotype like doxorubicin-resistant

MCF-7/Adr cells (93). More importantly, there is a significant

positive correlation between the expression levels of some

genes involved in MDR, such as MDR1 and multidrug

resistance-associated protein 1 (MRP1), and PKCy in acute

lymphoblastic leukemia (ALL) and acute myelogenous

leukemia (AML) patients (94). In fact, PKCy regulates the

MDR1 promoter activity in human breast carcinoma cells

(95). Therefore, PKCy could be of interest as a potential target

to overcome drug resistance in ALL and AML patients.

Summary and perspectives

Recent studies on PKCy have greatly improved our under-

standing of the selective function of this particular PKC iso-

form in T-cell activation, and established its important role in

the activation of mature T cells. Moreover, since PKCy coop-

erates with calcineurin to activate the IL-2 gene, it may repre-

sent a critical second signal for T-cell activation and IL-2

production. This raises the question of the potential link

between PKCy and the Ras signaling pathway, which is also

activated by phorbol esters via stimulation of the Ras activator

Ras-GRP (96). Indeed, these two signal mediators may be

functionally linked since both activate AP-1 and, furthermore,

dominant negative Ras interferes with AP-1 activation by PKCy(13). PKCy most likely integrates TCR/CD28 costimulatory

signals that are essential for activation of the NF-kB cascade

in T cells. The relatively selective expression and essential

function of PKCy in T-cell activation and survival suggest

that pharmacological or genetic strategies designed to select-

ively block the function of PKCy in cells may be therapeut-

ically useful in several potential scenarios. First, since TCR

engagement in the absence of CD28 costimulation can lead

Altman & Villalba � Protein kinase C-y (PKCy) location

60 Immunological Reviews 192/2003

to T-cell anergy (97, 98), inhibition of PKCy may ablate the

CD28 costimulatory signal and therefore promote anergy, a

possibility currently being tested by us. Second, PKCy inhibi-

tion could potentially abolish a T-cell survival signal and there-

fore promote the apoptosis of activated self-reactive T cells in

autoimmune diseases or, perhaps, synergize with apoptosis-

inducing regimens to enhance apoptosis of leukemic T cells or

other leukemias where the enzyme appears to be expressed

ectopically. However, in order to successfully apply these

approaches, it would be important to use regimens that tem-

porarily block PKCy function at a critical time without indu-

cing overt general immunosuppression.

Beyond this prospective use of PKCy as a drug target, there

remain several fundamental questions that need to be

answered. First, what is the mechanism that selectively recruits

PKCy to the cSMAC and what protein(s) or lipid(s) mediates

this recruitment? Second, what are the immediate physio-

logical substrates of PKCy and the critical intermediates in the

pathway leading from PKCy to NF-kB and AP-1 activation?

Third, does PKCy play a role in regulating the survival

of the normal T-cell pool in the immune system? Lastly, the

function and importance of PKCy during in vivo immune

responses and its potential role in immunological diseases, a

critical area, remain to be analyzed. The recent information

reviewed herein provides a solid foundation for future studies

that will undoubtedly answer these questions and uncover

additional details of the function and regulation of PKCy in

T cells.

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