148
Calcium Biochemistry
enzymes are encoded by different genes.
SkMLCK corresponds to a molecular mass
of 65 kDa, whereas the expression pat-
tern and genomic organization of sm/nm
MLCK is more complex. This is due to
alternative splicing that results in differ-
ent isoforms with signiFcantly different
molecular weights, ranging between 150
and 200 kDa owing to different lengths of
their amino termini.
To date, the regulatory myosin light
chain is the only established substrate
of MLCK. In smooth muscles, the Ca
2
+
-
dependent trigger of muscle contraction is
directly mediated by the phosphorylation
of the light chain by MLCK thus removing
the inhibition of the myosin ATPase.
In skeletal muscle, however, the acto-
myosin ATPase is regulated by binding
of Ca
2
+
to troponin C, the regulatory
calcium binding protein in the troponin
complex. A possible function of MLCK in
skeletal muscle is to modulate the rate
of enhancement and extent of isometric
contraction of the muscle.
The substrate speciFcity of MLCK is very
strict. Substrate recognition is basically de-
pendent on the appropriate location of
at least four basic residues over a more
extended region N-terminal of the phos-
phorylated serine residue. Calmodulin-
dependent enzymes are activated by dis-
rupting an autoinhibitory segment that
interacts with the catalytic core of the
enzyme and thus deinhibiting the en-
zyme. This concept was Frst developed
and veriFed for the cAMP-dependent pro-
tein kinase (PKA) that could be blocked by
an endogenous inhibitor (PKI) competing
with the substrate about the substrate bind-
ing site, thus serving as a pseudosubstrate.
MLCK is the CaM-dependent enzyme that
has been studied very thoroughly by Kemp
et al. to provide evidence for the existence
of a pseudosubstrate to serve as an au-
toinhibitory domain in MLCK. This view
gained further support by the Fnding that
controlled proteolysis at the C-terminal
end of MLCK generated a constitutively
active fragment independent of calmod-
ulin activation, that is, the autoinhibitory
domain was removed.
5.1.1.2.2
Multisubstrate
CaM
Kinases
┬▒our different enzymes of calmodulin-
dependent protein kinases fall into the
class of multifunctional or multisubstrate
kinases, CaMKI, CaMKII, CaMKIV, and
CaMKK, the activating kinase of CaMKI
and IV. This class can be subdivided
into CaMKII on one side and CaMKI,
CaMKIV,
and
CaMKK
on
the
other
for two reasons: (1) CaMKI, CaMKIV,
and CaMKK act as monomeric proteins,
whereas CaMKII is a multimeric enzyme
composed of up to 12 subunits; (2) CaMKI
and CaMKIV are activated by another
calmodulin-dependent protein kinase, that
is, by CaMKK. Whether CaMKK itself is
also activated by an upstream acting kinase
is not clear at the moment, but there
are indications that this indeed might be
the case. In contrast, CaMKII is activated
by autophosphorylation. Therefore, the
latter is discussed separately from CaMKI,
CaMKIV, and CaMKK.
CaMKII
Calmodulin-dependent
protein
kinase II (CaMKII) represents a family
of closely related protein kinases with
broad substrate speciFcity. This class of
protein kinases is involved in cellular
metabolism, neurotransmitter release, cy-
toskeletal structure, gene expression, cell
cycle control, and, more recently, in long-
term potentiation and depression (LTP and
LTD), functions that are thought to be in-
volved in memory and learning.
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