Diacylglycerol Signaling

122 E. Rozengurt
mechanisms, involving autophosphorylation or transphosphorylation, mediate the
phosphorylation of one or more residues within the activation loop leading to stabilization
of an active conformation of the catalytic residues (Johnson et al. 1996;
Oliver et al. 2007a). Many protein kinases that participate in signal transduction
pathways, including those in MAP-kinase cascades (Chang and Karin 2001; Ebisuya
et al. 2005; Johnson and Lapadat 2002), are regulated by transphosphorylation of the
activation loop mediated by a different upstream kinase. For example, Raf, the earliest
identified effector of Ras, transphosphorylates MEK on two key residues in its
activation loop, Ser 217 and Ser 221 , and thereby stimulates MEK activation (Marais and
Marshall 1996). It is also recognized that a substantial number of regulatory protein
kinases from different families mediate their own activation loop phosphorylation
promoting their catalytic activation. Examples of serine/threonine protein kinases
that mediate autoactivation include Aurora A (Eyers et al. 2003), Aurora B (Kelly
et al. 2007), CaMK II (Hudmon and Schulman 2002), Chk2 (Oliver et al. 2006),
DYRK (Lochhead et al. 2005), GSK-3 (Lochhead et al. 2006), JNK2 (Cui et al.
2005), Mps1 (Mattison et al. 2007; Kang et al. 2007), MTK1/MEKK4 (Miyake et al.
2007) and PAK (Buchwald et al. 2001). In many cases, autophosphorylation occurs
by intermolecular autophosphorylation (Oliver et al. 2007b).
Using 2-D tryptic phosphopeptide mapping of metabolically 32 P-labeled wild type
and mutant PKD forms, two key serine residues in the PKD activation loop, Ser 744 and
Ser 748 in mouse PKD (Fig. 7.1), were identified (Iglesias et al. 1998b; Waldron et al.
1999b). A PKD mutant with both sites altered to alanine was resistant to activation in
response to cell stimulation whereas mutation of Ser 744 and Ser 748 to glutamic acid
residues, to mimic phosphorylation, generated a constitutively active mutant PKD.
Single point mutants in which glutamic acid replaced Ser 744 and Ser 748 produced
partly activated kinases. The properties of these mutant forms of PKD were consistent
with a role of Ser 744 and Ser 748 in phosphorylation-dependent activation.
Using an antibody that recognizes PKD phosphorylated at Ser 744 /Ser 748 , and a
second antibody that detects predominantly PKD phosphorylated at Ser 748 , PKD activation
loop phosphorylation was demonstrated in response to regulatory peptides,
expression of heterotrimeric G proteins and oxidative stress in many cell types
(Zhukova et al. 2001a; Yuan et al. 2003; Waldron et al. 2004; Brandlin et al. 2002;
Storz et al. 2004b; Waldron et al. 2001). In line with the existence of a kinase cascade,
Ser 744 and Ser 748 also became rapidly phosphorylated in kinase-deficient forms of
PKD, indicating that PKD activation depends on transphosphorylation by an upstream
kinase (e.g., PKC) (Waldron et al. 2001). Although phosphorylation of other serine
(Matthews et al. 1999a; Iglesias et al. 1998b) and tyrosine (Waldron et al. 2004; Storz
and Toker 2003) residues are likely to play a role in PKD regulation, it is clear that
PKD phosphorylation at Ser 744 and Ser 748 is documented in multiple cell types, is triggered
by a vast array of stimuli and plays a critical role in PKD activation. However,
these initial experiments did not rule out that the activation loop residues, Ser 744 and
Ser 748 , are phosphorylated independently of each other or via an ordered mechanism
involving different isoforms of PKC or additional upstream kinases (see below).
Recent studies in vitro and in vivo examined further the role of PKCe as an upstream
kinase in the activation loop phosphorylation of PKD. When incubated in the presence