Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes.
The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.
Kulesza-Lipka
D
D
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Baines
I C
IC
Brzeska
H
H
Korn
E D
ED
D000048:Acanthamoeba; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009218:Myosins / Q000145:classification / Q000378:metabolism*; D009994:Osmolar Concentration; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D015800:Protozoan Proteins
D010750:Phosphoproteins; D015800:Protozoan Proteins; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D009218:Myosins