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A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3(-/-) cells.

ABSTRACT: Juvenile neuronal ceroid lipofuscinosis (JNCL) is a pediatric lysosomal storage disorder characterized by accumulation of autofluorescent storage material and neurodegeneration, which result from mutations in CLN3. The function of CLN3, a lysosomal membrane protein, is currently unknown. We report that CLN3 interacts with cytoskeleton-associated nonmuscle myosin-IIB. Both CLN3 and myosin-IIB are ubiquitously expressed, yet mutations in either produce dramatic consequences in the CNS such as neurodegeneration in JNCL patients and Cln3(-/-) mouse models, or developmental deficiencies in Myh10(-/-) mice, respectively. A scratch assay revealed a migration defect associated with Cln3(-/-) cells. Inhibition of nonmuscle myosin-II with blebbistatin in WT cells resulted in a phenotype that mimics the Cln3(-/-) migration defect. Moreover, inhibiting lysosome function by treating cells with chloroquine exacerbated the migration defect in Cln3(-/-). Cln3(-/-) cells traversing a transwell filter under gradient trophic factor conditions displayed altered migration, further linking lysosomal function and cell migration. The myosin-IIB distribution in Cln3(-/-) cells is elongated, indicating a cytoskeleton defect caused by the loss of CLN3. In summary, cells lacking CLN3 have defects that suggest altered myosin-IIB activity, supporting a functional and physical interaction between CLN3 and myosin-IIB. We propose that the migration defect in Cln3(-/-) results, in part, from the loss of the CLN3-myosin-IIB interaction.

SUBMITTER: Getty AL

PROVIDER: S-EPMC4124749 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3(-/-) cells.

Getty Amanda L AL Benedict Jared W JW Pearce David A DA

Experimental cell research 20100917 1

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a pediatric lysosomal storage disorder characterized by accumulation of autofluorescent storage material and neurodegeneration, which result from mutations in CLN3. The function of CLN3, a lysosomal membrane protein, is currently unknown. We report that CLN3 interacts with cytoskeleton-associated nonmuscle myosin-IIB. Both CLN3 and myosin-IIB are ubiquitously expressed, yet mutations in either produce dramatic consequences in the CNS such as neur ...[more]

PMID: 20850431

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Project description:Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.Significance of the studyPhosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.

Dataset Information

A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3(-/-) cells.

Publications

A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3(-/-) cells.

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