Phosphorylation and Dephosphorylation of the 20kDa Myosin Light Chain

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Myosin light chain kinase and myosin light chain phosphatase: Smooth muscle (as well as skeletal and cardiac muscle) contains myosin light chain kinase (MLCK), activated by Ca2+-calmodulin, the enzyme which transfers the terminal phosphate group of ATP to serine (and/or threonine) hydroxyl groups of phosphorylatable light chain (LC) according to the following reaction:

Dephosphorylation is brought about by smooth muscle myosin light chain phosphatase (MLCP) according to the following reaction:

The properties of MLCK are reviewed by Stull et al. (1996) and the properties of MLCP are reviewed by Hartshorne et al. (1998).

It is generally believed that LC phosphorylation-dephosphorylation controls the contraction-relaxation cycle of smooth muscle:

RELAXATION CONTRACTION

MLCK

in + Myosin,LC-OH + MgATPJ~ ++ Aetomyosin.LC-O-POj1" + MgADP + H+

MLCJ*

For a long time, research focused on the role of MLCK in smooth muscle contractility, but recently the interest shifted to MLCP. It turned out that MLCP is composed of three subunits: a catalytic subunit of 37-38-kDa of the type 1 phosphatase, a subunit of about 20-kDa whose function is not known, and a larger 110-130-kDa subunit that targets MLCP to myosin. The phosphatase activity of the catalytic subunit is low and it is enhanced significantly by addition of the targeting subunit. Upon phosphorylation of serine and threonine residues in the targeting subunit, its activating effect on the catalytic subunit is lost, and thereby the MLCP holoenzyme is inhibited.

Recent reports (Feng et al., 1999; Kaibuchi et al., 1999; Nagumo et al., 2000; Somlyo and Somlyo, 2000; Sward et al., 2000) indicate that in smooth muscle a Rho-regulated system of MLCP exists. Rho-kinase is the major player in this system, the enzyme phosphorylates the 130-kDa myosin binding subunit of MlCp and thereby inhibits MLCP activity. Due to the antagonism between MLCK and MLCP, inhibition of MLCP results in an increase in the phosphoryl content of LC with concomitant increase in muscle force. Under these conditions, submaximal Ca2+-levels are sufficient for maximal force, a phenomenon called increased Ca2+-sensitivity (Somlyo and Somlyo, 1994). Specific inhibitors for rho-kinase Y-27632 (Feng et al., 1999; Kaibuchi et al., 1999), and HA-1077 (Nagumo et al., 2000; Sward el al., 2000) are available.

MLCP activity can also be inhibited by a 17-kDa myosin phosphatase inhibitor protein, called CPI-17, (Kitazawa et al., 2000) , which inhibits the catalytic subunit of MLCP and the holoenzyme MLCP. Phosphorylation of CPI-17 at Thr38 increases its inhibitory potency 1000-fold. The solution NMR structure of CPI-17 has been determined.(Ohki et al., 2001), it forms a novel four-helix. Phosphorylation of Thr38 induces a conformational change involving displacement of one helix without significant movement of the other three helices. Rho-kinases and PKC are responsible for the phosphorylation of CPI-17.

A rich array of second messengers regulate MLCP activity under physiological and pathological conditions (Solaro, 2000) through phosphorylation of either the targeting subunit of MLCP or CPI-17.

Myosin light chain phosphorylation in intact smooth muscle: 32P-labeling of the muscle is a reliable method for such studies. When a dissected smooth muscle, e.g. artery or a uterine strip, is incubated at 37oC in physiological salt solution containing radioactive inorganic phosphate, the 32P permeates the plasma membrane and enters the intracellular space of the muscle. Through the oxidative phosphorylation mechanism the 32P incorporates into the terminal P group of ATP:

Transfer of the terminal 32P of ATP to LC-OH by MLCK (equation 1) yields the radioactive LC-O-32PO32- species that can be isolated and quantified. The isolation involves two-dimensional (2D) gel electrophoresis and the quantification requires measuring the specific radioactivity of the terminal P of ATP.

Smooth muscle contraction is correlated with LC phosphorylation (reviewed by Barany and Barany, 1996c). Fig. SM2 illustrates an experiment: Two carotid arteries were dissected from freshly killed pigs and labeled with 32P. One artery was contracted with KCl for 30 sec then frozen in liquid nitrogen, while the other artery was frozen in the resting state. The arteries were pulverized, washed with perchloric acid to precipitate the muscle proteins and remove 32P-containing phosphate metabolites from the muscle. The washed residue was neutralized with a NaOH solution then dissolved in sodium dodecyl sulfate (SDS). After centrifugation at high speed to remove insoluble particles, the protein content of the supernatant was determined and aliquots of 360 ^.g protein were subjected to 2D polyacrylamide gel electrophoresis. This procedure separates the proteins according to their charge (pH 4-6) in the first dimension and according to their size (SDS ) in the second dimension. After staining, the profile of the arterial proteins appeared, shown in the upper row of Fig. SM2. LC, is in the lower middle part of the gel, it contains multiple spots. The LC spots were scanned, the staining intensities are shown in the lower row of the Figure. The radioactive spots on the gel were detected by autoradiography, the middle row of Fig. SM2 shows the black spots on the film corresponding to the radioactive spots on the gel.

Visual inspection of the radioactive LC spots in the Figure shows much more radioactivity in LC from the contracting muscle (right) than from the resting muscle (left). One can calculate the incorporation of the 32P-phosphate into LC as follows. First one has to determine the specific radioactivity of the terminal P of ATP from the muscle. The ATP is in the perchloric acid extract of the frozen and pulverized muscle, described before, and Barany and Barany (1996c) describe the determination of the specific radioactivity. The next step is the determination of the radioactivity in LC: the gel spots are excised, digested with H2O2, and after the gel is dissolved, radioactivity (counts per minute) is measured. The extent of LC phosphorylation can be calculated from the radioactivity in the LC spots and in the terminal phosphate of ATP, from the total protein applied onto the gel, and from the LC content of the total protein (Barany and Barany, 1996c). Such a calculation shows that under conditions of Fig. SM2, the LC of the resting muscle contained 0.25 mol 32P-phosphate/mol LC, whereas the LC of the contracting muscle contained 0.70 mol. Thus, 0.45 mol 32P-phosphate was transferred by MLCK from the terminal phosphate of ADP32P to free LC-OH groups as the result of muscle contraction.

Fig. SM2. Light chain phosphorylation during smooth muscle contraction as studied by 2D gel electrophoresis. (Barany and Barany, 1996a, with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press). Left, 32P-labeled porcine carotid arterial muscle was frozen at rest. Right, 32P-labeled porcine carotid arterial muscle was frozen 30 sec after

100 mM KCl challenge. Upper panel shows the Coomassie blue staining pattern of the arterial proteins; middle panel shows the corresponding autoradiograms; bottom panel shows the corresponding densitometric scans of LC.

Isoforms of the 20-kDa myosin light chain: Protein isoforms have the same size but different charge. They are generated either by protein modification or genetic alteration. Protein phosphorylation is the physiological protein modification, because phosphorylation of a protein increases its negative charge. Thus, LC has at least two isoforms, a non-phosphorylated and a phosphorylated one. Genetic alteration changes the amino acid composition of a protein, thereby providing at least two isoforms. For instance, completely dephosphorylated LC exhibits two spots on 2D gels (Fig. SM3) with a percentage distribution of 85% and 15%, corresponding to the major and minor LC isoforms.

to a

Fig. SM3. Myosin light chain isoforms as analyzed by 2D gel electrophoresis. LC was dephosphorylated by homogenizing porcine carotid arteries in 150 mM NaCl and 1 mM EGTA, followed by incubation at 25oC for 2 hours. Top, stained gel, LC spots are numbered as 2 and 4, corresponding to their isoform number. Bottom, densitometric tracing of the LC spots.

If both major and minor LC are phosphorylated, that results in four isoforms (two non-phosphorylated and two phosphorylated). In Fig. SM2 on top level four LC isoforms are seen; on middle level, the autoradiograms reveal that three spots are radioactive. The non-radioactive, most basic spot, corresponds to the major isoform of LC (Fig. SM3).

Figure SM4 illustrates the formation of LC isoforms as a result of phosphorylation. The major isoform (LCa) when mono-phosphorylated (PLCa) moves into Spot 3, and when it is di-phosphorylated (2PLCa) moves into Spot 2. The same Spot 2 also contains the non-phosphorylated minor isoform (LCb), thus the comigration of the di-phosphorylated LC isoform with the minor isoform makes Spot 2 radioactive. This explains why out of the four LC spots three are phosphorylated. The mono-phosphorylated minor isoform (PLCb) moves into Spot 1, which is the most acidic spot.

Fig. SM4. Scheme for the explanation of four stained and three radioactive LC spots, shown on Fig. SM2. (Barany and Barany, 1996a, with permission from

Biochemistry of Smooth Muscle Contraction, 1996, Academic Press).

Fig. SM4. Scheme for the explanation of four stained and three radioactive LC spots, shown on Fig. SM2. (Barany and Barany, 1996a, with permission from

Biochemistry of Smooth Muscle Contraction, 1996, Academic Press).

The last row of Fig. SM2 shows the muscle contraction-induced changes in the staining intensity of LC spots. In the resting muscle the last peak, the non-phosphorylated major isoform contains as much as 63% of the total intensity, which decreases to 35% in the contracting, muscle. At the same time, the 23% intensity in the preceding peak of the resting muscle increases to 49% upon contraction. This is an example of a simple (but not sensitive) method to follow changes in LC phosphorylation during contraction, without using 32P-labeling of the muscle.

Phosphorylation site: The amino acid sequence of LC exhibits a similarity among LCs from various smooth muscles. Such a conservative sequence suggests a functional significance for the protein. The phosphorylation sites are located at the amino terminal part of the LC molecule, shown in Fig. SM5. Serine 19 is the site that is phosphorylated by mLck in the intact muscle. Threonine 18 is phosphorylated by MLCK rarely. Beside MLCK, protein kinase C (PKC) also phosphorylates LC; the sites involve Serine 1, Serine 2, and Threonine 9.

Fig. SM5. Phosphorylation sites of lc.

Two-dimensional tryptic peptide mapping: Phosphopeptide maps differentiate MLCK-catalyzed LC phosphorylation from that catalyzed by PKC (Erdodi et al., 1998). Fig. SM6 illustrates the experiment: With ADP32P as a substrate, pure LC was phosphorylated either by MLCK (middle panel), or PKC (right panel). Actomyosin that contains endogenous LC, MLCK, and PKC, was also phosphorylated (left panel). The 32P-LC was isolated by 2D gel electrophoresis, digested by trypsin, and the peptides were separated by 2D peptide mapping. The map of LC phosphorylated by MLCK exhibits four peptides: A, B, both containing serine residues, corresponding to the Ser-19 site, and C, D, both containing threonine, corresponding to the Thr-18 site. When LC is phosphorylated by PKC, the map exhibits two peptides: E, containing serine, corresponding to Ser-1 or Ser-2 site, and F, containing threonine, corresponding to theThr-9 site. When LC is phosphorylated in actomyosin, peptides characteristic for both MLCK and PKC phosphorylation are present.

Autoradiograms of 2D phosphopeptide maps of LC tryptic digests.

Autoradiograms of 2D phosphopeptide maps of LC tryptic digests.

Based on two-dimensional peptide mapping, one can identify the enzymes that phosphorylate LC during smooth muscle contraction (Fig. SM7). The phosphopeptide pattern of LC from K+-contracted artery (left part) is practically identical with that of LC{MLCK} pattern shown in Fig. SM6; thus, the conclusion is clear, MLCK is the main enzyme that phosphorylates LC during the K+-contraction. In contrast, when the muscle is contracted with phorbol dibutyrate (PDBu), an activator of PKC, the LC pattern (right part) resembles that of Actomyosin shown in Fig. SM6, thus, both enzymes, MLCK and PKC phosphorylate LC during the PDBu-contraction. One can determine the contribution of each enzyme to the phosphorylation, either by densitometry of the spots, or by counting the radioactivity in the spots, The results showed about 35% contribution by PKC.

K-contracted FDBu-treated

Fig. SM7. Phosphopeptide maps of LC from K+-contracted muscle versus PDBu-treated muscle.

The role of Ca2+ in light chain phosphorylation: As in skeletal muscle, Ca2+ also plays a central role in the contractility of smooth muscle. In skeletal muscle TN-C is the target of the myoplasmic Ca2+, whereas in smooth muscle Ca2+ activates MLCK. Actually, the Ca2+ complexed to calmodulin is the activator of the enzyme. In agreement with the in vitro studies, intact smooth muscle cease contracting when Ca2+ is omitted from the bathing solution, or when it is complexed with EGTA. Furthermore, inhibitors of calmodulin, such as trifluoperazine or chlorpromazine inhibit smooth muscle contraction.

In the resting muscle there is about 0.1 pM Ca2+, upon stimulation the Ca2+ concentration increases about 100-fold through electromechanical or pharmacomechanical coupling. It is conventional to use fluorescent indicators to follow changes in the intracellular Ca2+ concentration immediately after the stimulation and during the plateau of the mechanical activity. Large variations are reported, depending on the nature of the smooth muscle, the tissue preparation, or the drug used. However, all investigators agree that in order to elicit relaxation the Ca2+ level in the sarcoplasm must be returned near to the resting value. Two mechanisms participate in decreasing the Ca2+ level: 1) The plasma membrane Ca2+ transporting ATPase pumps Ca2+ from the inside into the extracellular space. 2) The sarco(endo)plasmic reticulum Ca2+ transporting ATPase pumps Ca2+ into the SR.

Stretch-induced light chain phosphorylation: As discussed before, smooth muscle can be stimulated electrically or by chemical agents. Here we describe the mechanochemical activation of smooth muscle. Stretching of arterial or uterine muscles induced light chain phosphorylation to the same extent as was observed in muscles contracted by K+ or norepinephrine (Barany and Barany, 1996c). Muscles which were stretched 1.6 times their resting length did not develop tension, but contracted normally when the stretch was released and the muscles were allowed to return to their rest length. Importantly, this contraction was spontaneous, indicating that the stretch-induced activation carries all the information necessary for normal contraction. Mobilization of Ca2+ was

Fig. SM7. Phosphopeptide maps of LC from K+-contracted muscle versus PDBu-treated muscle.

necessary for the stretch-induced light chain phosphorylation and contraction to occur. When EGTA (the strong Ca2+ complexing agent) was added to the muscle bath both the stretch-induced phosphorylation and the stretch-release-induced tension were inhibited; however, upon removal of EGTA by washings, both processes were fully restored. Treatment of the muscle with chlorpromazine (the calmodulin inhibitor) also abolished both the stretch-induced LC phosphorylation and the stretch-release-induced tension development. These results suggest the presence of mechanosensitive receptors in smooth muscle that are interacting with Ca2+ release channels in SR.

Further comments are warranted on the finding that 1.6 times stretched muscles, which are unable to contract (because there is no overlap between actin and myosin filaments), are able to fully phosphorylate their LC. Accordingly, smooth muscle contraction and LC phosphorylation are not coupled. Time course experiment also demonstrated that LC phosphorylation precedes tension development. Thus, LC phosphorylation plays a role in the activation process but not in the contraction per se. Furthermore, K+-contracted muscle maintains its tension for a prolonged time although its LC becomes dephosphorylated. This is another example for the lack of coupling between phosphate content of LC and contractility of muscle.

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