Insulin receptor tyrosyl kinase activity

Fig. 3. Artificial substrates for the insulin receptor tyrosyl kinase. Shows the Km values exhibited by basal and insulin-stimulated kinase activities together with the insulin-stimulated increase in Vmax for a variety of substrates. These include angiotensin and its modified derivative (VAL-5), also synthetic peptides of Glu Tyr and the so-called sarc -peptide, which bears the sequence around the tyrosyl autophosphorylation site of the sarc protein. Data are also given for a G-protein mixture of Gj/G0. These studies (referred to in the text) all employed soluble, purified insulin receptor preparations. No evidence has yet been presented for tyrosyl phosphorylation of substrates using isolated membrane preparations containing insulin receptors.

Tyrosyl phosphorylation of the solubilized insulin receptor also appears to affect its functioning per se, in that autophosphorylation leads to an increase in the receptor tyrosyl kinase activity expressed towards exogenous substrates [68]. As autophosphorylation occurs at multiple sites on the j8 subunit it will be necessary to determine which of these sites are of regulatory significance. Such studies require that results obtained using solubilized receptor preparations be compared with those using intact cells where tyrosyl phosphorylation of the receptor appears to be very small in comparison with the phosphorylation of the receptor on serine residues [69]. 

It remains to be seen whether the insulin receptor can activate Gins either directly or indirectly and whether the tyrosyl kinase activity of the receptor is involved. However, it is of interest that recently purified G and Gj have been shown to be capable of being phosphorylated by the insulin receptor tyrosyl kinase [113]. Such phosphorylation occurred on both the a and the /3 subunits. However, phosphorylation only occurred using the intact holomeric form of these G-proteins and not the activated, dissociated subunits. Other G-proteins may also be phosphorylated. However, whether any functional changes ensue and whether such reactions can occur in vivo. will have to be determined. 

There are distinct receptors for both IGF-I and IGF-II. The IGF-I receptor is similar in structure to that for the insulin receptor, having a disulphide bridge-linked subunit (a-j8)2 structure [49-52]. The a subunit has a molecular mass of 130 kDa which is capable of binding IGF-I. The 95 kDa j8 subunit of the IGF-I receptor, like that for the insulin receptor, exhibits a tyrosyl kinase activity. In marked contrast, however, the IGF-II receptor is a monomeric protein of molecular mass 220 kDa [53,54] with no known intrinsic activity. 

Half-maximal effects for insulin stimulation of the receptor tyrosyl kinase occurred at around 5-10 nM insulin and, from analysis of insulin specific-binding studies, it appears that there is a non-linear relationship between the stimulation of receptor autophosphorylation by insulin and receptor occupancy. In contrast, a linear relationship has been noted between steady-state insulin binding and the ability of solubilized receptor kinase preparations to phosphorylate various exogenous substrates. The kinetics of coupling between insulin binding and the ability to elicit kinase activation as regards both exogenous substrates and its autophosphorylative capability requires further study. 

Of particular interest are recent observations that the tyrosyl kinase activity of the insulin receptor can be attenuated by phosphorylation of the receptor on serine residues with cAMP-dependent kinase and protein kinase C [61,69,70]. The sites of phosphorylation for the action of these two kinases remain to be determined, however. 

In intact cells insulin has been shown to stimulate receptor autophosphorylation. However, much smaller amounts of phosphotyrosine were found compared to that seen using purified, solubilized receptor preparations and, indeed, the predominant phosphorylation actually occurred on serine residues [69,71]. It has been suggested that there may be an insulin-stimulated, phosphoseryl-specific kinase which is loosely associated with the receptor and is activated by the receptor tyrosyl kinase [61]. This might account for the insulin-stimulated phosphoseryl kinase activity observed in both intact cells and when using crude, solubilized receptor preparations. Such an activity might also provide a mechanism for insulin s ability to enhance serine phosphorylation on target proteins in plasma membranes and elsewhere in the cell [25,78]. Nevertheless, (auto)-phosphorylation of the insulin receptor on tyrosine residues has been shown to occur immediately upon receptor occupancy by insulin and to precede any phosphorylation on serine residues [69]. Indeed, it remains to be seen as to whether the seryl phosphorylation occurs as a direct insulin-stimulation event or mediated by other kinases (cAMP/C-kinase) as a consequence of insulin-stimulated autophosphorylation on tyrosyl residues. 

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