dues; the phosphodiester bonds; the thioether
bonds of lanthionines or lysinolanines in ukeratin; the cross-bridges in collagen and elastin,
formed by the oxidative deamination of lysine or
hydroxylysine (see Fig. 11.3, p. 379); and the bisand tertyrosines of resilins and other scleroproteins, which arise by the phenolic coupling of tyrosine residues (see Fig. 11.4, p.380). Most phosphorylations, acetylations and methylations probably have a regulatory function; glycosylation
serves not only for cell recognition but also for the
marking of proteins and the regulation of protein
degradation. Other modified protein amino acids
have special functions, e.g. y-carboxylglutamic
acid (Fig. 3.1) for Ca 2 + binding, or iodotyrosine in
the synthesis of the thyroid hormone [293]. The
variable post-translational glycosylation observed
in some homologous proteins of related species
raises the possibility that some modifications are
only the coincidental result of the activities of
available enzymes, and in particular cases have
no special functional significance.
3.1.2 Phosphorylation
and Dephosphorylation
The phosphorylation of the serine, threonine and
tyrosine residues of cellular proteins by specific
protein kinases plays a central role in the regulation of metabolism and differentiation. Several
protein kinases may often be found to act
sequentially in the form of a cascade; the effect of
regulatory signals is amplified and dispersed
through a network of protein phosphorylation
and dephosphorylation reactions, the complexity
of which is by no means fully understood [63].
The first protein kinases to be isolated and characterized were phosphorylase kinase in 1959 and
a cAMP-regulated protein kinase in 1968. In the
last 10 years, the number of known protein kinases has grown at an ever-increasing rate, particularly since it was discovered that the products of
many cellular oncogenes belong to the protein
tyrosine kinases. By 1987, there were already 50
protein serine/threonine kinases and 29 protein
tyrosine kinases known in mammals, a total of 18
protein kinases known in Drosophila, and 14 in
baker's yeast. It is possible that the mammalian
genome codes for up to 1000 different protein
kinases [37, 115].
All known protein kinases, including the oncogene products, show significant sequence similarity in their catalytic regions, and thus belong to
the same protein super-family [96]. Particular
3.1.2 Phosphorylation and Dephosphorylation
73
sequence motifs are always found and these can
be used for the identification of new protein kinases: at the N-terminus one finds the sequence
GXGXXG-, followed 15-20 amino acids further
on by a lysine residue; these amino acids belong
to the ATP binding site. At a distance 80-180
amino acids closer to the C-terminus, there is a
conservative region with the typical sequences
RDL, DFG and APE, and this facilitates a more
detailed classification of the protein kinases.
Between the motifs DFG and APE there is
always an autophosphorylatable amino acid; in
this region a tyrosine surrounded by acidic amino
acids distinguishes the protein as a protein tyrosine kinase [115]. Various types of protein kinase
occur both as particle-bound and free in the cytoplasm [40, 217]. The phosphorylation of cell proteins can be reversed by the action of protein
phosphatases. Although particular protein phosphatases are regulated, the majority are, in fact,
always active. Therefore, the degree of phosphorylation of individual proteins, and thus their
functional status, is mainly determined by the
regulated activity of the protein kinases.
Many protein kinases are regulated by secondary messengers and are hence closely involved in
signal transduction through the cell membrane.
The regulators of protein serine/threonine kinases are, on the hand, the cyclic nucleoside-3' ,5'monophosphates cAMP and cGMP and, on the
other hand, calcium ions in association with the
protein calmodulin, with phospholipids and diacylglycerins (protein kinase C), or also as free
ions. Recently, AMP-dependent protein kinases
have been discovered that apparently play an
important role in the regulation of lipid metabolism [25]. There are, however, a large number of
protein serine/threonine kinases for which no specific regulators are known. Protein tyrosine kinases function as membrane receptors for insulin
and various growth factors, and arise as products
of various retroviruses; they may also be found
not bound to membrane receptors in normal cells
[72, 115, 148]. In only a few instances are the
physiological substrates of the different protein
kinases identifiable. The majority of the protein
kinases show broad, overlapping substrate specificity in vitro, and it is often found that different
protein kinases phosphorylate the same protein
but at different positions. Many protein kinases
can phosphorylate themselves (autophosphorylation) and thus influence their own substrate affinity [252].
The protein kinases that are activated by cyclic
nucleoside monophosphates normally prefer one
bonds of lanthionines or lysinolanines in ukeratin; the cross-bridges in collagen and elastin,
formed by the oxidative deamination of lysine or
hydroxylysine (see Fig. 11.3, p. 379); and the bisand tertyrosines of resilins and other scleroproteins, which arise by the phenolic coupling of tyrosine residues (see Fig. 11.4, p.380). Most phosphorylations, acetylations and methylations probably have a regulatory function; glycosylation
serves not only for cell recognition but also for the
marking of proteins and the regulation of protein
degradation. Other modified protein amino acids
have special functions, e.g. y-carboxylglutamic
acid (Fig. 3.1) for Ca 2 + binding, or iodotyrosine in
the synthesis of the thyroid hormone [293]. The
variable post-translational glycosylation observed
in some homologous proteins of related species
raises the possibility that some modifications are
only the coincidental result of the activities of
available enzymes, and in particular cases have
no special functional significance.
3.1.2 Phosphorylation
and Dephosphorylation
The phosphorylation of the serine, threonine and
tyrosine residues of cellular proteins by specific
protein kinases plays a central role in the regulation of metabolism and differentiation. Several
protein kinases may often be found to act
sequentially in the form of a cascade; the effect of
regulatory signals is amplified and dispersed
through a network of protein phosphorylation
and dephosphorylation reactions, the complexity
of which is by no means fully understood [63].
The first protein kinases to be isolated and characterized were phosphorylase kinase in 1959 and
a cAMP-regulated protein kinase in 1968. In the
last 10 years, the number of known protein kinases has grown at an ever-increasing rate, particularly since it was discovered that the products of
many cellular oncogenes belong to the protein
tyrosine kinases. By 1987, there were already 50
protein serine/threonine kinases and 29 protein
tyrosine kinases known in mammals, a total of 18
protein kinases known in Drosophila, and 14 in
baker's yeast. It is possible that the mammalian
genome codes for up to 1000 different protein
kinases [37, 115].
All known protein kinases, including the oncogene products, show significant sequence similarity in their catalytic regions, and thus belong to
the same protein super-family [96]. Particular
3.1.2 Phosphorylation and Dephosphorylation
73
sequence motifs are always found and these can
be used for the identification of new protein kinases: at the N-terminus one finds the sequence
GXGXXG-, followed 15-20 amino acids further
on by a lysine residue; these amino acids belong
to the ATP binding site. At a distance 80-180
amino acids closer to the C-terminus, there is a
conservative region with the typical sequences
RDL, DFG and APE, and this facilitates a more
detailed classification of the protein kinases.
Between the motifs DFG and APE there is
always an autophosphorylatable amino acid; in
this region a tyrosine surrounded by acidic amino
acids distinguishes the protein as a protein tyrosine kinase [115]. Various types of protein kinase
occur both as particle-bound and free in the cytoplasm [40, 217]. The phosphorylation of cell proteins can be reversed by the action of protein
phosphatases. Although particular protein phosphatases are regulated, the majority are, in fact,
always active. Therefore, the degree of phosphorylation of individual proteins, and thus their
functional status, is mainly determined by the
regulated activity of the protein kinases.
Many protein kinases are regulated by secondary messengers and are hence closely involved in
signal transduction through the cell membrane.
The regulators of protein serine/threonine kinases are, on the hand, the cyclic nucleoside-3' ,5'monophosphates cAMP and cGMP and, on the
other hand, calcium ions in association with the
protein calmodulin, with phospholipids and diacylglycerins (protein kinase C), or also as free
ions. Recently, AMP-dependent protein kinases
have been discovered that apparently play an
important role in the regulation of lipid metabolism [25]. There are, however, a large number of
protein serine/threonine kinases for which no specific regulators are known. Protein tyrosine kinases function as membrane receptors for insulin
and various growth factors, and arise as products
of various retroviruses; they may also be found
not bound to membrane receptors in normal cells
[72, 115, 148]. In only a few instances are the
physiological substrates of the different protein
kinases identifiable. The majority of the protein
kinases show broad, overlapping substrate specificity in vitro, and it is often found that different
protein kinases phosphorylate the same protein
but at different positions. Many protein kinases
can phosphorylate themselves (autophosphorylation) and thus influence their own substrate affinity [252].
The protein kinases that are activated by cyclic
nucleoside monophosphates normally prefer one