Evidence against a role for alkaline phosphatase in the dephosphorylation of plasma membrane proteins: hypophosphatasia fibroblast study.
A major impasse to understanding the physiologic role(s) of alkaline phosphatase (ALP) is uncertainty as to its natural substrates. Various in vitro studies have led other investigators to suggest that ALP functions as a plasma membrane phosphoprotein phosphatase, consistent with our demonstration of ecto-topography of ALP in a variety of cell types. Thus, we compared the phosphorylation of plasma membrane proteins from control fibroblasts to those from profoundly ALP-deficient fibroblasts of hypophosphatasia patients. Fibroblasts from 3 controls and 3 hypophosphatasia patients (ALP activity < 4% of control) were biosynthetically labeled with 32Pi for 2 h. 32P incorporation into total trichloroacetic acid (TCA)-precipitable material was not significantly different in control and patient cells. Plasma membranes were prepared from these cells by hypotonic shock, solubilized, and subjected to two-dimensional (2-D) gel electrophoretic separation. Video densitometric analysis of silver-stained 2-D gels failed to reveal any consistent difference in the protein profile between patient vs. control fibroblasts (i.e., unique species, altered pls, or increased abundance). Autoradiography of individual 2-D gels demonstrated 63 plasma membrane phosphoproteins with molecular weights ranging from 15 to 152 kDa and predominantly acidic pls. Although several of these phosphoproteins appeared to have had donor-specific labeling, none was unique or especially abundant in the hypophosphatasia group. Thus, in ALP-deficient fibroblasts, normal incorporation of 32P into total cellular protein and into all identifiable plasma membrane phosphoproteins indicates that ALP does not modulate the phosphorylation of plasma membrane proteins.
Fedde
K N
KN
Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, Missouri 63110.
Michel
M P
MP
Whyte
M P
MP
D000469:Alkaline Phosphatase / Q000172:deficiency / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007014:Hypophosphatasia / Q000201:enzymology / Q000378:metabolism*; D007231:Infant, Newborn; D007526:Isoelectric Point; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D016622:Silver Staining
D008565:Membrane Proteins; D010750:Phosphoproteins; D000469:Alkaline Phosphatase