Protein phosphorylation: quantitative analysis in vivo and in intact cell systems.
Protein phosphorylation-dephosphorylation appears to be an essential component in the regulation of many cellular processes by hormones and drugs. This concept has developed primarily from in vitro biochemical studies in which various purified proteins have been phosphorylated and dephosphorylated by distinct protein kinases and phosphoprotein phosphatases. However, the more difficult, but essential, task of demonstrating the physiological occurrence of these reactions in intact tissue or cell preparations in many cases has not been undertaken in a quantitative manner. There are 4 basic approaches for assessing the extent of protein phosphorylation in vivo and in intact cell systems, each having particular advantages and disadvantages. These are summarized in Table 2. The applicability of any one procedure will be highly dependent upon the protein under investigation. For instance, chemical measurements of total protein-bound phosphate may provide only limited information for proteins which are phosphorylated at multiple sites but could be highly useful for those proteins such as glycogen phosphorylase which are phosphorylated at single sites. The relative ease and the high sensitivity of measuring 32P incorporation into proteins will tempt many investigators to rely heavily on this approach. It is a very powerful procedure, particularly for the initial identification of phosphoproteins, but ultimately quantitative conclusions regarding 32P incorporation must be corroborated by one or more of the other procedures. There is no simple, single experimental approach that may be used under all circumstances, but by integrating these procedures firm conclusions may be drawn regarding the physiological importance of phorphorylation of specific proteins.
D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D004592:Electrophoresis, Starch Gel; D007553:Isotope Labeling / Q000379:methods; D010710:Phosphates / Q000032:analysis*; D010749:Phosphoprotein Phosphatases / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis; D011817:Rabbits; D051381:Rats; D014020:Tissue Extracts / Q000032:analysis
D010710:Phosphates; D010750:Phosphoproteins; D014020:Tissue Extracts; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases