We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is re...

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 micr...

We cloned the full-length cDNA for the cytoplasmic myosin II regulatory light chain (RLC) from a stage 1-2 Xenopus oocyte library. The Xenopus RLC is 94% identical to the chicken smooth muscle myosin RLC. All of the protein kinase C and myosin light chain kinase phosphorylation sites are conserved. Using trifluoperazine [Trybus, K. M., Waller, G. S., & Chatman, T. A. (1994) J. Cell Biol. 124, 9...

We examined effects of a putative myosin light chain kinase inhibitor in the cerebral circulation in vivo. In anesthetized rats, diameter of basilar arteries was measured through a cranial window (control, 232 +/- 10 microns, mean +/- SEM). Vessel diameter was measured during topical application of agonists and antagonists. ML-7, which has been reported to compete with adenosine triphosphate fo...

We have purified myosin from human erythrocytes using methods similar to that for other cytoplasmic myosins with a yield of about 500 micrograms/100 ml of packed cells. It consists of a 200-kDa heavy chain and light chains of 26- and 19.5 kDa and therefore differs from the isozyme in platelets which has light chains of 20- and 15 kDa. At low ionic strength, the myosin forms short bipolar filame...

The Drosophila anterior-posterior (AP) axis is specified at mid-oogenesis when Par-1 kinase recruited to the posterior cortex of oocyte, where it polarises microtubule cytoskeleton define determinants, bicoid and oskar mRNAs localise. This polarity established in response an unknown signal from follicle cells, but how this occurs unclear. Here we show that myosin chaperone, Unc-45 Non-Muscle My...

Ca(2+) sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and i...

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect o...

p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting...