(Received for publication, March 24, 1995; and in revised form, August 18, 1995)
From the
A novel, 208-kDa myosin light chain kinase (MLCK) distinct from
smooth muscle and non-muscle MLCK has been identified by cross-reaction
to two antibodies raised against smooth muscle MLCK. Additional
antibodies directed against the amino and carboxyl termini of the
smooth muscle MLCK do not react with the 208-kDa MLCK, suggesting these
regions are distinct. 208-kDa MLCK phosphorylates 20-kDa myosin light
chains in a Ca/calmodulin-dependent manner,
consistent with it being a member of the MLCK family. Expression of
208-kDa MLCK and smooth muscle MLCK appears to be inversely regulated,
with 208-kDa MLCK being most abundant during early development and
declining at birth. In contrast, expression of smooth muscle MLCK is
relatively low early during development and increases to become the
predominant MLCK detected in all adult smooth and non-muscle tissues.
The developmental expression pattern of the 208-kDa MLCK suggests this
form be named, embryonic MLCK.
Myosin light chain kinases (MLCK) ()are
Ca
/calmodulin-regulated, actin and myosin binding,
Ser/Thr protein kinases (Kamm and Stull, 1985; Sellers and Pato, 1984).
In skeletal muscle, contraction is regulated by the troponin system and
phosphorylation of regulatory light chain by skeletal muscle MLCK has a
modulatory role in contraction-induced potentiation of isometric twitch
tension (Sweeney et al., 1993). In smooth muscle,
phosphorylation of the 20-kDa regulatory light chain of myosin by MLCK
is a well characterized event, important for the initiation of
contraction (Kamm and Stull, 1985). The role of myosin phosphorylation
by MLCK in non-muscle cells is not well characterized but correlates
with activities such as cell division, receptor capping, and platelet
or endothelial cell contraction (Kerrick and Bourguignon, 1984;
Kolodney and Wyslomerski, 1992; Wyslomerski and Lagunoff, 1990;
Holzapfel et al., 1983; Ehrlich et al., 1991;
Guiliano et al., 1992; Kolega and Taylor, 1993; Adelstein and
Conti, 1975; Garcia et al., 1995).
cDNAs representing
vertebrate smooth and non-muscle MLCKs have been sequenced and the
deduced amino acid sequences of the proteins compared (Gallagher et
al., 1991; Kobayashi et al., 1992; Shoemaker et
al., 1990; Olson et al., 1990). This comparison reveals a
high degree of sequence homology (>97% similarity) that extends
beyond the central catalytic core and calmodulin-binding autoinhibitory
region (Gallagher et al., 1991; Kobayashi et al.,
1992). The regions outside of the catalytic core and autoinhibitory
region are comprised of several class I and class II structural motifs
that are similar to those in the related family of giant muscle
proteins such as twitchin and titin (Olson et al., 1990;
Labeit et al., 1992). The amino terminus of the mammalian
MLCKs also contains a 175-residue insert not present in the avian
smooth and non-muscle MLCKs (Gallagher et al., 1991; Kobayashi et al., 1992). This insert is comprised of 15 tandem copies of
a 12-residue repeat of unknown function. Recently, an actin-binding
domain has been localized within the NH terminus between
residues 29 and 80 (Kanoh et al., 1993; Gallagher, 1994). The
extreme COOH terminus of smooth muscle MLCK is a highly acidic region
of unknown function that is also expressed as an independent protein,
telokin (Gallagher and Herring, 1991; Collinge et al., 1992;
Yoshikai and Ikebe, 1992). Telokin and by analogy, the COOH terminus of
smooth muscle MLCK have been proposed to be involved in myosin binding
and myosin filament assembly (Shirinsky et al., 1993).
Determination of molecular masses for smooth muscle MLCKs from
several species has shown that these proteins have a similar size
between 130 and 155 kDa (Gallagher et al., 1991). However, the
masses determined by SDS-PAGE are slightly larger than the masses
predicted by the deduced primary sequences (Gallagher et al.,
1991; Kobayashi et al., 1993). For example, the rabbit uterine
smooth muscle MLCK cDNA encodes a protein of 126 kDa while the
recombinant and tissue forms migrate at 152 kDa on SDS-PAGE. The amino
terminus of the chicken smooth muscle MLCK has been established by
direct sequencing of a CNBr subfragment obtained from the purified
protein (Faux et al., 1993). The six NH-terminal
residues (MDFRAN) identified correspond exactly to the first six
residues predicted by the 5` coding region for the chicken, rabbit, and
bovine cDNAs (Olson et al., 1990; Gallagher et al.,
1991; Kobayashi et al., 1993), suggesting that all of the
smooth muscle MLCKs have the same amino termini.
The non-muscle MLCK
is reported to be identical to smooth muscle isoforms except for the
presence of an additional 286-residues extending the NH terminus of the predicted protein (Shoemaker et al.,
1990). This additional sequence would increase the mass of the protein
by approximately 35 kDa (Shoemaker et al., 1990). Western
blotting of smooth and non-muscle tissues demonstrated that the major
form of MLCK expressed in these tissues has the same mass as the smooth
muscle MLCK. A larger protein corresponding to the non-muscle MLCK and
having an appropriate molecular mass (>165 kDa) as predicted by the
chicken embryo fibroblast cDNA has not been detected in any tissues
(Gallagher et al., 1991). Recently, Gibson and Higgins(1993)
suggested that the cDNA encoding the non-muscle MLCK may be incomplete
as additional class II structural motifs appear to be present in the
5`-untranslated region of the reported cDNA.
The failure to detect a
larger mass MLCK in non-muscle tissues may be because this protein is
expressed at low levels or because it is expressed only in specific
non-muscle tissues. It is also possible that this cDNA, isolated from
primary embryonic fibroblast cells, represents a form of MLCK that is
developmentally regulated and its expression may not be detectable in
adult tissues. For these reasons, we have examined MLCK expression in
embryonic and adult tissues and in cultured cells by Western blotting.
These studies reveal the existence of a 208-kDa protein, named
embryonic MLCK because its expression can be detected in early
embryonic tissues, stem cells, and in proliferating cultured cells.
208-kDa protein is a Ca/calmodulin-dependent MLCK
that is structurally distinct both from the smooth muscle MLCK and the
protein predicted by the chicken non-muscle MLCK cDNA.
Figure 1: Western analysis of myosin light chain kinase detected in tissues and cells. A, analysis of rat tissues and rodent cell lines. Amounts of total protein represented in each lane are indicated above figure. B, detection of MLCKs in smooth and non-muscle tissues. Between 50-100 µg (smooth muscle tissues) and 100-175 µg (non-muscle tissues) total protein are represented in each lane. Blot is overexposed to detect expression of 208-kDa protein. C, analysis of muscle-derived and non-muscle cultured cell lines. Cell extracts prepared either from non-muscle (REF, COS, CEF (chicken embryo fibroblast), HUVEC (human umbilical endothelial), or muscle-derived cell lines (A10, AT1, and AT2) were examined. 50 µg of total protein is represented in all lanes except HUVEC, AT1 and AT2 (25 µg each). Positions of molecular weight markers and MLCKs are indicated at the left side of the figure.
Smooth muscle MLCK (130-155 kDa) can be detected in most cultured cell lines, with relatively higher expression levels being detected in muscle-derived cell lines such as A10 smooth muscle, the AT-1 and AT-2 cardiac muscle lines (Fig. 1C). Although not visible in Fig. 1C, 130-155-kDa smooth muscle MLCKs can be detected in chicken embryo fibroblast, COS, and human umbilical endothelial cells, when greater amounts of total protein are analyzed. However, the predominant immunoreactive protein in most cells lines is 208-kDa MLCK. Only two cell lines, REF (Fig. 1C) and undifferentiated murine embryonic stem cells, lack any detectable level of smooth muscle MLCK expression using the available reagents.
Figure 2:
Immunological relationship of 208-kDa
protein and smooth muscle MLCK. A, Western immunoblotting
using four antibodies to detect MLCK in cell extracts from A10 and REF
cell lines. Each lane represents 50 µg of total protein. B, ligand blotting to detect immunoprecipitated embryonic MLCK
by biotinylated calmodulin in the presence of Ca.
Embryonic MLCK was immunoprecipitated from REF cells using anti-Repeat
antibody (Repeat IP) or preimmune serum (Pre-immune IP) and
protein A-Sepharose, separated by SDS-PAGE, transferred to
nitrocellulose, and reacted with 20 nM biotinylated calmodulin
in the presence of Ca
. C, Western
immunoblotting using unadsorbed(-) or preadsorbed (+)
anti-smMLCK or anti-Repeat antibodies to detect MLCK in cell extracts
from A10 (left lane) and REF cell lines (right lane).
Positions of molecular weight markers and MLCKs are indicated at the
left side of the figures.
To determine if the anti-smMLCK antibody recognizes epitopes in addition to the repeated sequence present in mammalian MLCK, this serum was preadsorbed using the repeat peptide. A solid phase binding assay was performed to confirm that the antibodies to the repeated region had been completely adsorbed (data not shown). As shown in Fig. 2C, the preadsorbed serum recognizes both 136-kDa smooth muscle MLCK and 208-kDa protein, confirming that at least some antigenic epitopes in addition to the repeated region exist between the smooth muscle MLCK and the 208-kDa protein. A control Western blotting experiment using preadsorbed anti-Repeat antibody showed a loss of detection of both 136-kDa rat smooth muscle MLCK and 208-kDa protein in both A10 and REF cell extracts.
Figure 3:
Myosin light chain phosphorylation by
immunoprecipitated 208-kDa embryonic MLCK. A, representative
curves showing Ca/calmodulin-dependent incorporation
of
P into myosin light chains following
immunoprecipitation of 208-kDa protein using anti-Repeat antibody from
2 volumes of cell extract (200 and 100 µl). Comparable low rates of
incorporation were obtained using protein immunoprecipitated by
anti-Repeat antibody in the presence of EGTA (200 µl) or using
preimmune serum (P.I.). B, autoradiogram showing
incorporation of
P into 20-kDa regulatory light chains.
Assays were terminated by addition of SDS-PAGE sample buffer. Proteins
were separated by electrophoresis (15% SDS-PAGE) and incorporation of
P into 20-kDa regulatory light chain detected by
autoradiography of the dried gel. Also shown are samples using
preimmune serum to immunoprecipitate REF cell
extracts.
Examination of
proteins labeled by incorporation of P during the assay
revealed that the only significant radiolabeled protein was the myosin
regulatory light chain (Fig. 3B). Autoradiography
showed that only those reactions containing Ca
/CaM
and extracts immunoprecipitated by anti-Repeat antibody (Fig. 3B) or anti-smMLCK antibody (data not shown)
resulted in significant incorporation of
P into light
chains.
Figure 4:
Northern analysis of mRNAs in rat cells
and tissues. Northern blot analysis of RNA isolated from rat uterine
tissue (15 µg of total cellular RNA) and rat A10 cells (5 µg of
poly(A)) following fractionation through a 1.2%
agarose gel. The blot was probed with
P-labeled cDNA probe
corresponding to nucleotides 714-3705 (residues 136-1134) of the
full-length rabbit uterine smooth muscle MLCK. The positions of the RNA
molecular weight standard are shown in kilobases on the left. The blot
was exposed for 48 h at -70 °C. Longer exposure of the blot
reveals the presence of an 8.7-kb mRNA in rat
tissues.
Figure 5:
Developmental expression of MLCKs in
murine tissues and stem cells. Immunoblots in panel A were
reacted with anti-Repeat antibody which has the highest sensitivity for
208-kDa embryonic MLCK. The blots were stripped and reacted with
anti-NH-terminal antibody which has the highest sensitivity
for 130-kDa murine smooth muscle MLCK (panel B). Embryonic
stem cells, undifferentiated or differentiated by replating embryoid
bodies were extracted as described under ``Materials and
Methods.'' Whole embryos, embryonic heart, or liver tissues were
collected at the times (days post-fertilization) indicated above the
blots. E, embryonic; Neo, neonatal. Each lane
represents 50 µg of total cellular protein. Positions of molecular
weight markers (left) and MLCKs (right) are
indicated.
A 208-kDa MLCK has been identified as a new member of the
MLCK family based upon its immunological and biochemical properties.
The 208-kDa MLCK is detected in extracts of cultured cells and tissues
by two polyclonal antibodies specific for smooth muscle MLCK
(anti-smMLCK and anti-Repeat antibody). In activity assays, 208-kDa
MLCK phosphorylates purified myosin regulatory light chains in a manner
consistent with the Ca/calmodulin-dependent
properties of all previously characterized eucaryotic MLCKs (Stull et al., 1991; Kamm and Stull, 1985). As the 208-kDa MLCK is
expressed in undifferentiated embryonic stem cells and early embryonic
tissues, we suggest the name embryonic MLCK.
The lack of
immunoreactivity of 208-kDa embryonic MLCK with two additional
antibodies specific for smooth muscle MLCK
(anti-NH-terminal and anti-COOH-terminal) suggests that the
amino and carboxyl termini of this molecule differ from the smooth
muscle MLCK, which is detected by both these antibodies. This result
also distinguishes 208-kDa embryonic MLCK from the predicted protein
encoded by the chicken embryo fibroblast non-muscle MLCK cDNA
(Shoemaker et al., 1990).
The developmental expression pattern of 208-kDa embryonic MLCK is of interest, as many actomyosin-dependent events occur in proliferating, migrating cells that are distinct from those occurring in differentiated, non-proliferative tissues. The temporal expression pattern of 208-kDa embryonic MLCK compared to 130-kDa murine smooth muscle MLCK shows that this form is expressed at high levels early during development in embryonic tissues. Expression of 208-kDa embryonic MLCK declines at birth to low or undetectable levels in most adult tissues, although in some tissues such as liver down-regulation of 208-kDa embryonic MLCK is not as dramatic. The decline in expression of embryonic MLCK in cardiac tissues is generally coincident with cessation of cardiomyocyte proliferation and terminal differentiation which occurs shortly following birth in rodents (Rumyantsev, 1991). In liver, a less dramatic decline in expression of embryonic MLCK occurs, possibly reflecting the high regenerative capacity of this tissue. The coincidental expression of 208-kDa embryonic MLCK during early development, in undifferentiated embryonic stem cells and cultured cell lines suggests that the functional role of the 208-kDa embryonic MLCK may be to regulate the activity of early developmental or non-muscle forms of myosin. As non-muscle myosin heavy chain isoforms are expressed in many cultured cell lines and early during development, expression of 208-kDa embryonic MLCK in proliferating cultured cells or in differentiating tissues is consistent with the proposal that this form of MLCK has a unique regulatory function for these myosin isoforms. If this is true, then it is plausible that a family of MLCKs exists, each being specialized to regulate the activities either of specific myosin isoforms or specialized myosin motor activities such as assembly/disassembly, contraction, or cytokinesis.
Collectively, the immunological and biochemical data presented in this report identify the presence of a previously undetected form of MLCK. We suggest this form of MLCK be called embryonic MLCK because of its detection in undifferentiated stem cells and early embryonic tissues. Expression of embryonic MLCK is down-regulated following birth in most adult tissues, with the predominant form of MLCK in adult tissues being smooth muscle MLCK. It is now apparent the expression of smooth muscle MLCK is not tissue-specific and the expression patterns of both forms of MLCK during development suggest that embryonic MLCK and smooth muscle MLCK are inversely regulated. The pattern of immunoreactivity of embryonic MLCK clearly shows that this form of MLCK is related to but clearly distinct from both the smooth muscle MLCK and the predicted non-muscle MLCK.