Stimulus-induced phosphorylation of PKC h at the C-terminal
hydrophobic-motif in human T lymphocytesq,qq
Michael Freeleya, Yuri Volkovb,c, Dermot Kelleherb,c, Aideen Longa,*
aDepartment of Biochemistry, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland
bDepartment of Clinical Medicine, Trinity College, St. James?s Hospital, Dublin, Ireland
cDublin Molecular Medicine Centre, St. James?s Hospital, Dublin 8, Ireland
Received 13 June 2005
Available online 1 July 2005
Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three
conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the
C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC bIand d are con-
stitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC h is constitutively phosphorylated
at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation
at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the
membrane. Studies on the stimulus-induced phosphorylation of PKC h demonstrate that an upstream kinase activity involving a
conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR
pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane
translocation, suggesting that this site may control other aspects of PKC h signalling.
? 2005 Elsevier Inc. All rights reserved.
Keywords: Protein kinase C h; Hydrophobic-motif; T lymphocyte
Protein kinase C (PKC) is a family of phospholipid-
dependent serine/threonine kinases that play key roles
in many of the signalling pathways that control cellular
growth, proliferation, differentiation, and cell death .
PKC isoforms are divided into three groups based on
their N-terminal regulatory domain structure and
requirements for lipid second messengers. Conventional
PKCs (cPKCs) comprise the a, bI/bII, and c isoforms,
and are regulated in a Ca2+and diacylglycerol (DAG)-
dependent manner and also require the membrane-local-
ised co-factor phosphatidylserine (PS) for optimal
activity. The novel PKCs (nPKC) comprise the d, e, g,
h, and l isoforms, and function in a Ca2+-independent
manner but require DAG and PS for activity. Finally,
the atypical PKC family (aPKC), comprising f and
i/k, functions in a PS-dependent, but Ca2+and DAG-in-
dependent, manner. DAG and Ca2+ions accumulate in
the cell following receptor-mediated activation of phos-
pholipase C (PLC), which hydrolyses the inositol
phospholipid PtdIns(4,5)P2into DAG (which activates
0006-291X/$ - see front matter ? 2005 Elsevier Inc. All rights reserved.
qThis work was supported by the Royal College of Surgeons in
Ireland Research Committee, The Health Research Board of Ireland
and Enterprise Ireland.
qqAbbreviations: aPKC, atypical PKC; cPKC, conventional PKC;
DAG, diacylglycerol; FBS, foetal bovine serum; IL-2, interleukin-2;
ILK, integrin-linked kinase; LFA-1, lymphocyte function-associated
antigen-1; MAPKAPK, mitogen-activated protein kinase-activated
kinase-2; mTOR, mammalian target of rapamycin; NF-jB, nuclear
factor of jB; NP40, nonidet P40; nPKC, novel PKC; PBS, phosphate-
buffered saline; PDK-1, phosphoinositide-dependent protein kinase-1;
PI3-kinase, phosphatidylinositide-30-OH kinase; PKA, protein kinase
A; PKB, protein kinase B; PKC, protein kinase C; PMA, phorbol 12-
myristate 13-acetate; PS, phosphatidylserine.
*Corresponding author. Fax: +353 1 402 2467.
E-mail address: firstname.lastname@example.org (A. Long).
Biochemical and Biophysical Research Communications 334 (2005) 619–630
PKC) and Ins(1,4,5)P3, the production of which leads to
the release of Ca2+from intracellular stores. Stimulus-
induced increases in the levels of DAG (and Ca2+for
cPKCs) recruit these enzymes to membranes. Binding
of DAG to the regulatory domain of PKC induces a
conformational change in the enzyme, resulting in
expulsion of the autoinhibitory pseudosubstrate from
the catalytic cleft, thus permitting substrate binding
and phosphorylation .
PKC activity is controlled by a complex set of pro-
cesses that includes regulation by lipid second messen-
gers (e.g., DAG), binding to anchoring proteins, and
phosphorylation of the enzyme itself . The cPKC
and nPKCs contain three conserved phosphorylatable
serine/threonine residues located at the carboxyl-termi-
nal catalytic domain that are essential for catalytic activ-
ity, intracellular distribution, and stability . These
sites are known as the activation-loop, the turn-motif,
and the C-terminal hydrophobic-motif (the aPKCs con-
tain a phosphorylatable activation-loop and turn-motif
residue but possess a negatively charged glutamic-acid
at the hydrophobic-motif site, perhaps mimicking a con-
stitutively phosphorylated state). These phosphorylation
sites are also conserved on many other kinases termed
ABC kinases (representing PKA,1PKB, PKC, and other
kinase families such as p70 and p90 ribosomal S6 ki-
nases). While PDK-1 phosphorylates the activation-
loop of many ABC kinases in vivo (including PKCs)
, the mechanism of phosphorylation at the other
two residues, in particular at the hydrophobic-motif site,
is unclear. For many of these kinases, conflicting evi-
dence in the literature suggests that these enzymes either
autophosphorylate at the hydrophobic-motif  or that
a heterologous kinase activity (putatively termed PDK-
2) phosphorylates this site (see Discussion).
T lymphocytes predominantly express PKC family
members a, bI, d, e, g, h, l, and f [5,8]. PKC h is the only
member of the T cell-expressed PKCs that localises to
the immunological synapse  and activates important
transcription factors such as NF-jB . Our group has
demonstrated that the expression of PKC bIis crucial
for integrin-mediated migration of T lymphocytes 
and we (and others) have also shown that the expression
of this enzyme is required for the secretion (but not pro-
duction) of the cytokine interleukin-2 (IL-2) in response
to T cell activation [10,11]. The phosphorylation status
of endogenous PKC isoforms at these sites in response
to T cell receptor activation stimuli has not been ana-
lysed in detail.
In the present study, we have characterised the phos-
phorylation status of PKC isoforms bI, d, and h at these
sites in resting and activated T cells. Unlike PKC bIand
d, PMA or anti-CD3/CD28 stimulation induces hydro-
phobic-motif phosphorylation (Ser695) of PKC h. We
also provide evidence that an upstream kinase activity
involving the PI3-kinase pathway and cPKC isoforms,
rather than autophosphorylation or the rapamycin-sen-
sitive mTOR pathway, is responsible for the phosphor-
ylation of this site on the enzyme. These findings
suggest that cPKCs may act as PDK-2-like enzymes
for PKC h and regulate its function in T lymphocytes.
Although hydrophobic-motif phosphorylation of PKC
h coincides with translocation of the enzyme to the
membrane, phosphorylation of this site does not appear
to be important for membrane localisation.
Materials and methods
Reagents and antibodies. The Jurkat leukaemic T cell line and the
monoclonal anti-CD3 producing hybridoma cell line (OKT-3) were
obtained from the American Type Culture Collection (Manassas, VA).
RPMI-1640, foetal bovine serum (FBS), penicillin, streptomycin,
L-glutamine, cell culture grade phosphate-buffered saline (PBS), and
cell culture flasks were obtained from Gibco BRL (Life Technologies,
Paisley, UK). Sterile six-well plates were obtained from Nunc (Ros-
kilde, Denmark). Go6976, staurosporine, cyclosporine A, and rapa-
mycin were obtained from Calbiochem (Nottingham, UK). LY294002
was obtained from Alexis (Nottingham, UK). Protein G–Sepharose
beads were obtained from Amersham Biosciences (Bucks, UK).
Polyvinylidene fluoride (PVDF) membrane was obtained from Pall
Gelman Laboratories (Ann Arbor, MI). Nonidet P40 (NP40) and
EDTA were obtained from B.D.H. chemical and laboratory supplies
(Poole, UK). All other chemicals (including rottlerin and wortmannin)
were from Sigma (St. Louis, MO). Rabbit anti-mouse IgG was ob-
tained from Dako A/S (Denmark). Mitogenic antibody to human
CD28 was obtained from Ancell (Bayport, MN). Monoclonal anti-
bodies to PKC b were obtained from Seikagaku (Tokyo, Japan), BD
Biosciences (Oxford, UK), and Zymed (San Francisco, CA) (the anti-
PKC b antibody from Zymed was used for immunoprecipitation,
whilst the anti-PKC b antibodies from Seikagaku and BD Biosciences
were used for immunoblotting). Antibodies to total PKC d were ob-
tained from BD Biosciences and Santa Cruz Biotechnology (Santa
Cruz, CA) (the anti-PKC d antibody from BD Biosciences was used for
immunoprecipitation, whilst the Santa Cruz antibody was used for
immunoblotting). The anti-PKC h antibody was obtained from BD
Biosciences and was used for both immunoprecipitation and immu-
noblotting. The anti-LFA-1 antibody was obtained from Serotec
(Oxford, UK). The following antibodies were obtained from Cell
Signalling Technology (Beverly, MA): phospho-PKC h (p-Thr538)
antibody (recognises the phosphorylated activation-loop), phospho-
PKC d (p-Ser643) antibody (recognises the phosphorylated turn-motif
of PKC d and h), phospho-PKC a/bII(p-Thr638/Thr641) antibody
(recognises the phosphorylated turn-motif of PKC a and b), and
phospho-pan PKC (recognises the phosphorylated C-terminal hydro-
phobic-motif of all PKC isoforms). Goat anti-mouse IgG-HRP, goat
anti-rabbit IgG-HRP, anti-ERK 1/2 (total), and the Phototope HRP
detection kit were also obtained from Cell Signalling Technology.
The P500 antibody that recognises the phosphorylated activation-
loop threonine residue of all PKC isoforms was generated by Joanne
Johnson in the Newton laboratory and was generously provided by
Alexandra Newton (University of California, US). The specificity of
this antibody has been characterised elsewhere and was used at a 1/
1000 dilution . Antisera generated against the phosphorylated
forms of PKC h at Ser676 and Ser695 (phosphorylated turn-motif and
hydrophobic-motif of PKC h, respectively) and their non-phosphory-
1PKA does not possess a hydrophobic-motif residue as the protein
sequence terminates immediately prior to the hydrophobic-motif
M. Freeley et al. / Biochemical and Biophysical Research Communications 334 (2005) 619–630
In summary, we have shown that T cell activation
induces hydrophobic-motif phosphorylation and mem-
brane translocation of PKC h. This stimulus-induced
phosphorylation of PKC h at this site is independent
of the rapamycin-sensitive mTOR pathway but is sensi-
tive to inhibitors of PI3-kinase and a conventional PKC
inhibitor. This suggests that the PI3-kinase pathway and
cPKCs (such as PKC a) control PKC h phosphorylation
at this site and regulate its enzymatic activity and intra-
cellular function in T cells. It appears therefore that
cPKCs function as PDK-2-like enzymes for this critical
mediator of T cell signalling. Hydrophobic-motif phos-
phorylation is not critical for membrane translocation
however, which suggests that this site controls other
important aspects of kinase function.
We thank Professor Alexandra Newton for the kind
gift of the P500 phospho-activation-loop PKC antibody
and Dr. Stephen Shaw for kindly donating the phospho-
Ser676/Ser695 antisera to PKC h.
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