the N-lobe. The R-spine is completed by the N-lobe residues L95 (alpha C) and
Leu106 located in the β4-strand. The formation of the R-spine is indicative of an
active conformation of αC and the DFG-motif and is therefore a structural hallmark
of the kinase active state.
In agreement with the important structural role of the C-spine, mutation of
Leu173 to alanine in PKA and the analogues residue in CDK2 results in kinase
inactivation [27], and R-spine mutations have been detected oncogenic mutants
resulting in kinase activation in particular in the so-called C-spine gatekeeper
position that controls access to an extended ATP pocket in kinases with small
amino acids in this position [28–30].
3 Mechanism of Kinase Activation
Activation of protein kinase is tightly controlled by a multitude of regulatory
mechanisms. A key regulatory structural element is the activation segment, which
is typically 20–40 residues long and consists of the DFG magnesium ion-binding
motif, a short β-strand (β9), the actual activation loop and the P+1 loop. The
activation segment shows considerable conformational diversity between two
invariable anchor points at the N- and C-terminus of this segment: the DFG motif
and the P+1 loop linking the activation segment to helix αEF [19].
For kinases requiring phosphorylation for activity, the unphosphorylated
activation segment is usually unstructured or assumes an inactive conformation
[31, 32]. Phosphorylation of the activation segment at a serine, threonine or tyrosine
residue located typically about 11 residues N-terminal to the APE sequence
motif stabilizes the A-loop. (Fig. 3). The role of further phosphorylation sites
is however less clear. Phosphorylation at a secondary site is required for the activity
of extracellular signal-regulated kinase 2 (ERK2) [33]. In contrast, introducing
a second phosphorylation site in glycogen synthase kinase 3 (GSK3) increases
the catalytic activity only moderately, and modulation of substrate selectivity
has been suggested as a potential role of these phosphorylation events [34].
There are two mechanisms of activation segment phosphorylation – autoactivation
and phosphorylation by a kinase acting upstream in a signal transduction pathway.
Autoactivation is poorly understood as it requires that an inactive (unphosphorylated)
kinase activates itself by trans-phosphorylation. Several models have been
established including increasing local concentration by ligand-induced receptor dimerization [35, 36] or oligomerization in the cytoplasm as observed for CAMK2 [37–
39]. Some intramolecular mechanisms have also been described. For instance, the -
dual-specificity tyrosine phosphorylation-regulated kinase phosphorylates
its own activation segment on a tyrosine residue. After this initial intramolecular
phosphorylation event, this kinase trans-phosphorylates exclusively substrates on
serine and threonine residues, whereas tyrosine autophosphorylation in GSK3
requires the presence of chaperonins [40, 41].
6
S. Röhm et al.
Leu106 located in the β4-strand. The formation of the R-spine is indicative of an
active conformation of αC and the DFG-motif and is therefore a structural hallmark
of the kinase active state.
In agreement with the important structural role of the C-spine, mutation of
Leu173 to alanine in PKA and the analogues residue in CDK2 results in kinase
inactivation [27], and R-spine mutations have been detected oncogenic mutants
resulting in kinase activation in particular in the so-called C-spine gatekeeper
position that controls access to an extended ATP pocket in kinases with small
amino acids in this position [28–30].
3 Mechanism of Kinase Activation
Activation of protein kinase is tightly controlled by a multitude of regulatory
mechanisms. A key regulatory structural element is the activation segment, which
is typically 20–40 residues long and consists of the DFG magnesium ion-binding
motif, a short β-strand (β9), the actual activation loop and the P+1 loop. The
activation segment shows considerable conformational diversity between two
invariable anchor points at the N- and C-terminus of this segment: the DFG motif
and the P+1 loop linking the activation segment to helix αEF [19].
For kinases requiring phosphorylation for activity, the unphosphorylated
activation segment is usually unstructured or assumes an inactive conformation
[31, 32]. Phosphorylation of the activation segment at a serine, threonine or tyrosine
residue located typically about 11 residues N-terminal to the APE sequence
motif stabilizes the A-loop. (Fig. 3). The role of further phosphorylation sites
is however less clear. Phosphorylation at a secondary site is required for the activity
of extracellular signal-regulated kinase 2 (ERK2) [33]. In contrast, introducing
a second phosphorylation site in glycogen synthase kinase 3 (GSK3) increases
the catalytic activity only moderately, and modulation of substrate selectivity
has been suggested as a potential role of these phosphorylation events [34].
There are two mechanisms of activation segment phosphorylation – autoactivation
and phosphorylation by a kinase acting upstream in a signal transduction pathway.
Autoactivation is poorly understood as it requires that an inactive (unphosphorylated)
kinase activates itself by trans-phosphorylation. Several models have been
established including increasing local concentration by ligand-induced receptor dimerization [35, 36] or oligomerization in the cytoplasm as observed for CAMK2 [37–
39]. Some intramolecular mechanisms have also been described. For instance, the -
dual-specificity tyrosine phosphorylation-regulated kinase phosphorylates
its own activation segment on a tyrosine residue. After this initial intramolecular
phosphorylation event, this kinase trans-phosphorylates exclusively substrates on
serine and threonine residues, whereas tyrosine autophosphorylation in GSK3
requires the presence of chaperonins [40, 41].
6
S. Röhm et al.