CST Guide:

Section I: Research Areas
chapter 02: Signaling
Activators and
Inhibitors of PKC
#9841 Bisindolylmaleimide I,
Hydrochloride
Potent inhibitor of
PKC family members
#11916 Chelerythrine Chloride
Cell-permeable inhibitor of
PKC; activates SAPK/JNK
and p38-MAPK; induces
apoptosis in some cell lines
#12060 Gö6976
Potent inhibitor of
calcium-dependent
PKC family members
#9995 Ionomycin, Calcium Salt
Calcium ionophore;
activates calcium-/calmodulin-dependent
kinase and
calcium-dependent PKCs
#9953 Staurosporine
Very broad, ATP-competitive
protein kinase inhbitor (PKC,
PKA, Src, CaM kinase, etc.)
#4174 TPA (12-O-Tetradecanoylphorbol-13-Acetate)
Cell permeable, potent
activator of PKC; also used
to activate MAPK.
Calcium, cAMP, and Lipid Signaling
Calcium is a critical regulator of a diverse set of cellular functions, and maintaining calcium homeostasis
is a highly regulated mechanism involving numerous proteins and hormones. Calcium ion channels and
pumps regulate calcium entry and exit from cells in response to a stimulus. Inside the cell, calcium binding
proteins regulate local calcium concentrations and help transduce calcium signals. Enzymes such as
PKC and PLC respond to elevated levels of calcium and transmit signals to downstream signaling nodes.
Protein Kinase C (PKC)
Protein kinase C (PKC) family members regulate numerous cellular responses including gene expression,
protein secretion, cell proliferation, and the inflammatory response. The basic protein structure
includes an N-terminal regulatory region connected to a C-terminal kinase domain by a hinge region.
PKC enzymes contain an auto-inhibitory pseudosubstrate domain that binds a catalytic domain
sequence to inhibit kinase activity. Differences among PKC regulatory regions allow for variable second
messenger binding and are the basis for the division of the PKC family into 3 broad groups. Conventional
PKC enzymes (cPKC; isoforms PKCα, PKCβ, and PKCγ) contain functional C1 and C2 regulatory
domains; cPKC enzyme activation requires binding of diacylglycerol (DAG) and a phospholipid to the
C1 domain, and calcium binding to the C2 domain. Novel PKC enzymes (nPKC; isoforms PKCδ, PKCε,
PKCη, and PKCθ) also require DAG binding for activation but contain a novel C2 domain that does not
act as a calcium sensor. Distantly related protein kinase D proteins are often associated with novel PKC
enzymes as they respond to DAG but not calcium stimulation. Atypical enzymes (aPKC; isoforms PKCζ
and PKCι/λ) contain a nonfunctional C1 domain and lack a C2 domain, requiring no second messenger
binding for aPKC activation.
The enzyme PDK1 or a close relative is responsible for PKC activation. Control of PKC activity is regulated
through three distinct phosphorylation events. Phosphorylation occurs in vivo at Thr500 in the
activation loop, at Thr641 through autophosphorylation, and at the C-terminal hydrophobic site Ser660.
Phospholipase C (PLC)
Phosphoinositide-specific phospholipase C (PLC) plays a significant role in transmembrane signaling.
In response to extracellular stimuli such as hormones, growth factors, and neurotransmitters, PLC
hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate the secondary messengers inositol
1,4,5-triphosphate (IP3) and diacylglycerol (DAG). At least four families of PLCs have been identified:
PLCβ, PLCγ, PLCδ and PLCε. PLC activity is largely regulated by phosphorylation. For example,
phosphorylation of PLCβ3 at Ser1105 by PKA or PKC inhibits activity, whereas phosphorylation of PLCγ
at Tyr 771, 783, and 1245 by both receptor (EGFR) and nonreceptor tyrosine kinases (Syk) results
in activation. In addition, members of the PLCβ subfamily are activated by the α- or β/γ-subunits of
heterotrimeric G-proteins and play an important role in GPCR signaling cascades.
Growth factor stimulation results in phosphorylation of PLCγ1 at Tyr783.
A
Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb #14008: Confocal IF analysis of A-431 cells, untreated (A), treated with hEGF #8916
(100 ng/ml, 5 min) (B), or treated with hEGF #8916 (100 ng/ml, 5 min) and λ phosphatase (C), using #14008 (green). Blue pseudocolor
= DRAQ5 ® #4084 (fluorescent DNA dye). WB analysis of extracts from NIH/3T3 cells, untreated (-) or stimulated with hPDGF-BB #8912
(5 min; +), and from A-431 cells, untreated (-) or stimulated with hEGF #8916 (5 min; +) (D), using #14008 (upper) and β-Actin (D6A8)
Rabbit mAb #8457 (lower).
B
C
D
kDa
200
140
100
80
60
50
40
30
60
50
40
30
1 2
– + – –
– – – +
Phospho-
PLCγ1
(Tyr783)
β-Actin
hPDGF-BB
hEGF
Akt/PKB
p70S6K
PDK1
PKCζ/λ PKCδ PKCθ PKCµ PKCα/βII
Growth Factors,
Insulin, etc.
PI3K
Activated
By TPA
Receptor
Calcium Binding Proteins
Calcium is a critical second messenger for intracellular signaling pathways that regulate a diverse
range of biological functions such as cell growth, motility, contractility, membrane trafficking, neurotransmitter
release, apoptosis, and differentiation. Low molecular weight calcium binding proteins
such as calmodulin, calbindin, S-100, paravalbumin, and troponin C bind to calcium via EF hand
domains and help transduce intracellular signals by activating downstream target proteins or act as
calcium buffering proteins to regulate local calcium concentrations. For example, the calcium binding
protein calbindin is expressed in discrete neuronal populations within the CNS, including Purkinje cells,
and is thought to act as an intracellular calcium buffering protein. Calbindin is highly expressed in
neurons during migration and differentiation.
MARCKs
Calcium binding protein calbindin is expressed
in discrete neuronal populations within the CNS.
Phorbol ester TPA
activates PKCδ, resulting
in phosphorylation
at Thr505.
kDa
105
76
57
Phospho-
PKCδ
(Thr505)
0 15 30 60 120 240 TPA (min)
Phospho-PKCδ (Thr505) Antibody
#9374: WB analysis of extracts from
U-937 cells, untreated or TPA-treated
(0.2 µM), using #9374.
PKCθ is a novel protein kinase C predominantly expressed in T cells.
CD3
10 4
10 3
10 2
10 1
10 0 10 1 10 2 10 3 10 4
10 0
10 0 10 1 10 2 10 3 10 4
Rabbit Isotype Control
PKCθ
PKCθ (E1I7Y) Rabbit mAb
#13643: Flow cytometric analysis
of mouse splenocytes using Rabbit
(DA1E) mAb IgG XP ® Isotype Control
#3900 (left) and #13643 (right).
Splenocytes were co-stained with
anti-CD3 APC and the Anti-rabbit
IgG (H+L), F(ab’) 2 Fragment (PE
Conjugate) #8885 was used as a
secondary antibody.
Calbindin (D1I4Q) XP ® Rabbit mAb
#13176: Confocal IF analysis of normal rat
brain (A) using #13176 (green) and GFAP
(GA5) Mouse mAb #3670 (red). Blue pseudocolor
= DRAQ5 ® #4084 (fluorescent DNA
dye). IHC analysis of paraffin-embedded rat
brain (B) using #13176.
A
B
80 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstcalcium 81