Purification and Characterization of a Kinase-associated, Myofibrillar Smooth Muscle Myosin Light Chain Phosphatase Possessing a Calmodulin-targeting Subunit

Sobieszek, Apolinary; Babiychuk, Eduard B.; Ortner, Birgit; Borkowski, Jacek

doi:10.1074/jbc.272.11.7027

Cited by 20 publications

(32 citation statements)

References 39 publications

(30 reference statements)

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“…Thus, some analogy to our myofibrillar 37-and 67-kDa phosphatase is apparent. The possible relationship between these "old" phosphatases and our myofibrillar phosphatase was discussed in the preceding paper (9). Therefore, we will restrict the present discussion to the more recent myofibrillar MLCPase of Alessi et al (10).…”

Section: Fig 1 Separation Of Mlckase and Mlcpase Activities During mentioning

confidence: 99%

“…However, the phosphatase, which was not bound to the CaM affinity column, was usually free of the kinase activity. The fraction that is bound to, and eluted from, CaM affinity columns in the presence of Ca 2ϩ is dealt with in the preceding paper (9). The MLCKase preparations eluted from CaM affinity column in the absence of Ca 2ϩ are heavily contaminated by the phosphatase, even when the columns are washed with 0.36 M salt.…”

Section: Purification Of the Mlckase And Mlcpasementioning

confidence: 99%

“…In our new approach, the phosphatase was purified by CaM affinity chromatography and was shown to be composed of 37-kDa catalytic and 67-kDa targeting subunits (9). The only other myofibrillar phosphatase known so far was purified and partly characterized by Alessi et al (Ref.…”

Section: ؉mentioning

confidence: 99%

“…The fractions containing MLCKase and MLCPase activities eluted together as a relatively wide peak of about 350 kDa. These fractions were further purified on QSepharose FF or DEAE-Sepharose 6B-CL columns as described in the preceding paper (9). Although this purification step made it possible to identify the SDS-PAGE bands corresponding to MLCPase catalytic (PC) and targeting (PT) subunits, a satisfactory separation of the kinase and the phosphatase was not obtained.…”

Section: Purification Of the Mlckase And Mlcpasementioning

confidence: 99%

“…Coupling of ReLC to CNBr-activated Sepharose 4B and thiophosphorylation of ReLC affinity columns as well as their elutions were performed as described (9). SDS-PAGE was performed in 7.5-15 or 9 -18% gradient acrylamide minislab gels by the procedure of Matsudaira and Burgess (16) in the buffer system of Laemmli (17) with some improvements as described (18).…”

Section: Chemicals and Protein Preparations-[␥-mentioning

confidence: 99%

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Purification and Characterization of a Smooth Muscle Myosin Light Chain Kinase-Phosphatase Complex

Sobieszek

Borkowski

Babiychuk

1997

Journal of Biological Chemistry

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We show that a myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) forms a multienzyme complex with myosin light chain kinase (MLCKase). The stability of the complex was indicated by the copurification of MLCKase and MLCPase activities through multiple steps that included myofibril preparation, gel filtration chromatography, cation (SPSepharose BB) and anion (Q-Sepharose FF) exchange chromatography, and affinity purification on calmodulin and on thiophosphorylated regulatory light chain columns. In addition, the purified complex eluted as a single peak from a final gel filtration column in the presence of calmodulin (CaM). Because a similar MLCPase is present in varying amounts in standard preparations of both MLCKase and myosin filaments, we have named it a kinase-and myosin-associated protein phosphatase (KAMPPase).The KAMPPase multienzyme complex was composed of a 37-kDa catalytic ( Phosphorylation of myosin by myosin light chain kinase (MLCKase) 1 represents the key activation step leading to contraction of smooth muscle (for reviews, see Refs. 1-4). Relaxation or inactivation of myosin is accomplished by a myosin light chain phosphatase (MLCPase) that has a controversial identification and subunit composition (see Ref. 5). In numerous previous studies (for references, see Ref. 6), many types of cytosolic MLCPases have been purified exhibiting different specific activities toward phosphorylated myosin or isolated myosin regulatory light chain (ReLC). A common feature of all of these phosphatases seems to be the presence of not only a catalytic (PC) subunit of about 35-38 kDa but also another subunit in the range of 55-72 kDa. The function of the latter subunit has not been established. Initial attempts to classify these serine/threonine phosphatases were not very successful (7), and it appears that smooth muscle MLCPases could be either the PP1 or the PP2A type.Several years after our initial report on the first myofibrillar MLCPase (8), we and others again turned our attention to MLCPases from smooth muscle. In our new approach, the phosphatase was purified by CaM affinity chromatography and was shown to be composed of 37-kDa catalytic and 67-kDa targeting subunits (9). The only other myofibrillar phosphatase known so far was purified and partly characterized by Alessi et al. (Ref. 10; see also Refs. 11 and 12). It is composed of three subunits: a 37-kDa catalytic subunit and two regulatory subunits of 130 and 20 kDa. Although the sequence of all three subunits has been determined, the role of the regulatory subunits is not understood (see Ref. 5). In this report, we describe further results on our myofibrillar smooth muscle protein phosphatase, which is closely associated with MLCKase and myosin filaments and is called, therefore, a kinase-and myosin-associated protein phosphatase (KAMPPase). We show for the first time that this association results in a functional multienzyme complex between these two key regulatory enzymes of smooth muscle.

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Section: Fig 1 Separation Of Mlckase and Mlcpase Activities During mentioning

confidence: 99%

Section: Purification Of the Mlckase And Mlcpasementioning

confidence: 99%

Section: ؉mentioning

confidence: 99%

Section: Purification Of the Mlckase And Mlcpasementioning

confidence: 99%

Section: Chemicals and Protein Preparations-[␥-mentioning

confidence: 99%

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Purification and Characterization of a Smooth Muscle Myosin Light Chain Kinase-Phosphatase Complex

Sobieszek

Borkowski

Babiychuk

1997

Journal of Biological Chemistry

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Immunochemical characterization of myosin-specific phosphatase 1 regulatory subunits in bovine endothelium

2000

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We have previously shown that myosin-specific phosphatase 1 (PPase 1) activity is critical for maintaining endothelial cell barrier function (Verin et al. [1995] Am. J. Physiol. 269:L99-L108). To further characterize myosin-specific PPase 1 in endothelium, we generated antibodies specific to published sequences of the myosin-associated PPase 1 regulatory subunit (M110) from smooth muscle. Peptide antigens were designed based upon consensus sequences for a single ankyrin repeat (ANK 110) and a leucine zipper motif region (LZ 110), which represents putative sites for binding the PPase 1 catalytic subunit (CS1) and myosin, respectively. Our initial study demonstrated that each antibody immunoprecipitated 2 proteins with an apparent Mr of 110 and 70 kD on SDS-PAGE. The CS1delta isoform, which appeared to be characteristic for the myosin-specific phosphatase, was co-immunoprecipitated under non-denaturing conditions with ANK110 and LZ110 as was actin, myosin, and myosin light chain kinase (MLCK). Similarly, immunoprecipitation with specific anti-myosin or anti-MLCK antibodies under the same conditions, followed by immunostaining with either LZ110 or ANK110 revealed the same 110 and 70 kD protein bands. The 70 kD protein (p70) was immunoreactive with ANK 110 and LZ 110, was complexed with myosin and MLCK, and was detected in non-denaturing M110 immunoprecipitates. Consistent with these results, endothelial cell fractionation demonstrates the presence of p70 in both cytoskeletal and myosin-enriched fractions, but not in the myosin-depleted (cytosolic) fractions. These data suggest that endothelial cells may exhibit two distinct myosin-specific PPase 1 regulatory subunits which share certain structural features with the M110 regulatory subunit from smooth muscle and which are tightly associated with myosin and MLCK in a functional complex.

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Regulation of cross-bridge cycling by Ca2+ in smooth muscle

Arner

Pfitzer

1999

Reviews of Physiology Biochemistry and Pharmacology, Volume 134

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Purification and Characterization of a Kinase-associated, Myofibrillar Smooth Muscle Myosin Light Chain Phosphatase Possessing a Calmodulin-targeting Subunit

Cited by 20 publications

References 39 publications

Purification and Characterization of a Smooth Muscle Myosin Light Chain Kinase-Phosphatase Complex

Purification and Characterization of a Smooth Muscle Myosin Light Chain Kinase-Phosphatase Complex

Immunochemical characterization of myosin-specific phosphatase 1 regulatory subunits in bovine endothelium

Regulation of cross-bridge cycling by Ca2+ in smooth muscle

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