NONRECEPTOR CYTOSOLIC PROTEIN-TYROSINE KINASES FROM VARIOUS RAT-TISSUES

Adipocytic-cytosolic non-receptor protein tyrosine kinase (CytPTK) whe n activated can substitute for the insulin receptor tyrosine kinase (I nsRTK), in manifesting several insulin effects in insulin-receptor ind ependent fashion. Our aims here were to utilize PolyGlu(4)Tyr, a good experimental exogenous substrate for protein tyrosine kinases (PTKs) i n general, for studying qualitative and quantitative parameters of Cyt PTKs extracted from different tissue cytosols. At the same time, we wo uld search for a unique specific marker specifically characterizing Cy tPTKs. High speed supernatants of spleen, thymus, smooth muscle, lung and kidney were found to be rich in CytPTK activities. Their specific activities being 6- to 13-fold that of liver or adipose cytosols. Brai n, testis and adrenal cytosols were an intermediate source of CytPTK a ctivity, whereas CytPTK activity of heart and skeletal muscle was low. It was also evaluated that the capacity of the cytosol to phosphoryla te PolyGlu,Tyr is 15-50% that of the non-stimulated Triton X-100 extra ctable plasma membrane PTKs. Fractionation of the cytosols on superose 12 column revealed several CytPTKs within the same tissue, their peak s ranging between 30 and 450 kDa. Immunoblotting analysis showed Fyn a nd Lyn were present in most tissue cytosols. Upon immunoprecipitation, however, with anti-Fyn or anti-Lyn, negligible amounts (< 2%) of the total cellular CytPTK were precipitated. Thus, these general markers o f CytPTKs comprise only a minor proportion of the total intracellular PolyGlu(4)Tyr phosphorylating capacity. To see whether a specific mark er for CytPTK could be detected, we also examined the requirement of C ytPTKs for divalent ions, their preferred phosphate donor and their se nsitivity to inhibition by known PTK inhibitors. We found that the ord er of reactivity with divalent cations was Co2(+)> Mn2+ > Mg2+, while Zn2+ and Ca2+ did not support CytPTK activity. The best phosphate dono r was ATP (ED(50)=5 mu M), but other nucleoside 5-phosphates could sub stitute for ATP at high concentrations. With respect to these paramete rs, no basic difference exists between cytosolic and plasma-membrane P TKs. The PTK inhibitors, genestein and quercetin, inhibited both cytos olic and membranal PTKs at micromolar concentrations. In contrast, sta urosporine was a potent inhibitor of CytPTKs (IC50 5-20 nM) and a poor inhibitor of membranal PTKs (IC50 10-40 mu M). One of the conclusions we can draw from this study is that tissue cytosols contain PolyGlu(4 )Tyr phosphorylating capacity in quantities greater than previously as sumed and that the low level of phosphotyrosine found in cells is not the result of limited intracellular levels of CytPTKs.