Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.
Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.
Fazio
M J
MJ
Department of Biological Sciences, University of Illinois at Chicago 60607, USA.
Da Silva
A C
AC
Rosiere
T K
TK
Bouck
G B
GB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D047388:Casein Kinases; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D005056:Euglena gracilis / Q000201:enzymology* / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010206:Papain; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*
D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; C076623:articulin; D000255:Adenosine Triphosphate; D006493:Heparin; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; D010206:Papain