Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Gao, Ying; Kawano, Kazufumi; Yoshiyama, Shinji; Kawamichi, Hozumi; Wang, Xiaoming; Nakamura, Akio; Kohama, Kazuhiro

doi:10.1016/s0006-291x(03)00690-9

Cited by 11 publications

(9 citation statements)

References 27 publications

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“…Alternatively, MLCK may bypass MLC phosphorylation and act directly on myosin. This possibility is supported by findings that MLCK can bind directly to myosin II and stimulate its ATPase activity (14,54). Conceivably, MLCK may influence NKCC activity directly as reported for a Ca 2ϩ channel (50).…”

Section: Discussionmentioning

confidence: 57%

“…MLCK has been shown to alter ion transport through both MLC phosphorylation-dependent and -independent mechanisms (50). Furthermore, whether MLC phosphorylation or merely myosin activity is involved in transporter regulation remains questionable, because the latter can be regulated in a phosphorylation-independent manner as well (14,54). To approach these issues, we targeted MLC phosphorylation per se rather than MLCK activity.…”

Section: Differential Effects Of K525a On Basal Activity and Hyperosmmentioning

confidence: 99%

“…MLCK has been shown to influence myosin ATPase activity in a MLC phosphorylation-independent fash- ion (14,54) by directly binding to the myosin heavy chain and stimulating myosin ATPase activity. Furthermore, while an increase in myosin phosphorylation is not the signal for enhanced NKCC activity, a basal level of myosin activity may be a necessary permissive factor for the hypertonic response.…”

Section: Effect Of Mlc Mutants On Osmotic Stress-induced Mlc Phosphormentioning

confidence: 99%

“…Even if such a relationship exists, however, it remains to be determined whether an increase in myosin phosphorylation is in fact a signal for the osmotic activation of NKCC or whether a basal level of MLC phosphorylation (or myosin ATPase activity) is merely a permissive factor necessary for the maximal osmotic stimulation of the cotransporter. Finally, MLCK-mediated regulation does not necessarily occur through myosin phosphorylation, because MLCK can activate myosin ATPase activity without phosphorylation (14,54), and MLCK has been reported to act on transporters in a myosin-independent manner (50).…”

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confidence: 99%

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Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na⁺-K⁺-2Cl⁻ cotransporter?

Ciano-Oliveira¹,

Lodyga²,

Fan³

et al. 2005

American Journal of Physiology-Cell Physiology

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Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na+-K+-2Cl− cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl− depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl− depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC.

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Section: Discussionmentioning

confidence: 57%

Section: Differential Effects Of K525a On Basal Activity and Hyperosmmentioning

confidence: 99%

Section: Effect Of Mlc Mutants On Osmotic Stress-induced Mlc Phosphormentioning

confidence: 99%

mentioning

confidence: 99%

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Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na⁺-K⁺-2Cl⁻ cotransporter?

Ciano-Oliveira¹,

Lodyga²,

Fan³

et al. 2005

American Journal of Physiology-Cell Physiology

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“…The role of the nonkinase activity was characterized mostly in vitro (9,32). We hypothesized that the regulation of the interaction between actin and myosin by the nonkinase activity should play an important role in vivo, i.e., in living VSMCs.…”

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confidence: 99%

Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine

Wang

Nakamura²,

Matsumoto³

et al. 2009

American Journal of Physiology-Heart and Circulatory Physiology

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The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration.

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Survey of the year 2003 commercial optical biosensor literature

2005

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In the year 2003 there was a 17% increase in the number of publications citing work performed using optical biosensor technology compared with the previous year. We collated the 962 total papers for 2003, identified the geographical regions where the work was performed, highlighted the instrument types on which it was carried out, and segregated the papers by biological system. In this overview, we spotlight 13 papers that should be on everyone's 'must read' list for 2003 and provide examples of how to identify and interpret high-quality biosensor data. Although we still find that the literature is replete with poorly performed experiments, over-interpreted results and a general lack of understanding of data analysis, we are optimistic that these shortcomings will be addressed as biosensor technology continues to mature.

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Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Cited by 11 publications

References 27 publications

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na⁺-K⁺-2Cl⁻ cotransporter?

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na⁺-K⁺-2Cl⁻ cotransporter?

Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine

Survey of the year 2003 commercial optical biosensor literature

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Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain

Cited by 11 publications

References 27 publications

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl− cotransporter?

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl− cotransporter?

Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine

Survey of the year 2003 commercial optical biosensor literature

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Product

Resources

About

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na⁺-K⁺-2Cl⁻ cotransporter?

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na⁺-K⁺-2Cl⁻ cotransporter?