The N-terminus of the long MLCK induces a disruption in normal spindle morphology and metaphase arrest.
We have shown previously that only the long myosin light chain kinase (MLCK), which is the predominant MLCK isoform expressed in nonmuscle cells, localizes to the cleavage furrow. To further examine the in vivo localization of the long MLCK in HeLa cells and the mechanisms responsible for kinase targeting during the cell cycle, we examined the distribution of the endogenous kinase and constructed green fluorescent protein (GFP) fusions of long HeLa MLCK truncations. A GFP fusion containing the N-terminal IgG domain and the five DXR motifs localized to stress fibers during interphase and the cleavage furrow during mitosis. Although individual fusions of the five DXRs and IgG domain both independently localized to stress fibers, only the five DXRs demonstrated a cortical localization in mitotic cells. Thus, robust targeting of the long MLCK to the cleavage furrow required the five DXRs and additional sequences from the IgG domain. Expression of the IgG domain alone or with five DXRs increased the number of multinucleate cells tenfold, whereas expression of the five DXRs or GFP had no effect. Furthermore, expression of the IgG domain alone or with five DXRs disrupted normal spindle morphology during mitosis. Extended astral microtubules and increased bundling of kinetochore microtubules, and spindle pole fragmentation were detected in mitotic cells. These microtubule defects were associated with abnormalities in metaphase chromosome alignment and a subsequent metaphase arrest caused by activation of the spindle assembly checkpoint at the kinetochores of mono-oriented chromosomes. Together, these results suggest that MLCK has an unexpected regulatory function during mitosis.
Dulyaninova
Natalya G
NG
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Patskovsky
Yury V
YV
Bresnick
Anne R
AR
D000199:Actins / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002869:Chromosome Aberrations; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism; D008677:Metaphase; D018715:Microscopy, Video; D008870:Microtubules / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism / Q000502:physiology*
D000199:Actins; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007527:Isoenzymes; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase