Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic β-cells

Abstract

From the evidence presented in this review there is no doubt that protein kinases play vital roles in the regulation of insulin secretion from pancreatic β-cells. Most of the published studies concur with a model for insulin secretion (Figure 6) in which Ca2+-regulated protein kinases are involved in the initiation of insulin-secretory responses by the physiologically important nutrient secretagogues, although other Ca2+- sensitive response elements may also be required for a full secretory response. In this consensus model kinases from the PKA and PKC families are vitally important for the modulation of secretory responses by receptor- operated, nonnutrient secretagogues, but their involvement in nutrient- induced insulin secretion is still a matter of debate. Definitive evidence is not yet available about the involvement of other kinase transduction pathways in the regulation of insulin secretion, including the MAPK cascade and those operating through tyrosine kinases/phosphatases. Part of the uncertainty about the roles of protein kinases in β-cells can be attributed to the expression of multiple isoforms of these enzymes, and this highlights an important question for future studies: what is the functional significance of protein kinase isoforms in β-cells? The answer may be complex and may differ for each class of protein kinase. For example, the different isoforms of CaMK II expressed in β-cells (Section II.A) are subject to the same mechanism of regulation by Ca2+/CaM and, so far as is known, are found in the same locations within the cell, although the truncated γ-isoform (63) may have access to areas denied the full-length enzymes. In this case the existence of similar isoforms from independent genes may simply reflect a fail-safe adaptation to ensure the expression of active and regulatable CaMK activity in the event of deleterious mutations of the gene encoding one isoform. The isoforms of PKA are also subject to the same mechanism of regulation of enzymic activity, in this case by the availability of cAMP, but the subcellular localization of PKA activity is determined by the isoform of the regulatory subunit within the holoenzyme (Section II.B). The expression of PKA isoforms, or at least those of the regulatory subunit, may therefore exist to subserve specific functions, and the regulation of activity, substrate specificity, and cellular function may be determined as much by cellular location as by the isoform structure. The PKC family of isoforms differs from CaMK and PKA in that subgroups of isoforms are subject to different mechanisms of regulation and may be confined to different intracellular compartments (Section II.C). One explanation for this diversity is that the subgroups of PKC isoforms (conventional, novel, atypical) have evolved to subserve specific and different functions within β-cells, in which case experimental studies of PKC function should be directed toward individual isoforms, or at least isoform subgroups, to produce interpretable information. Substantial insights into protein kinase function in β-cells will be dependent upon the development of novel methods for the detection, activation, and inhibition of individual protein kinase isoforms. Despite the overwhelming evidence of kinase involvement in the secretory process, we are still largely ignorant of the identity and functions of the kinase substrates (Section III.B), and this ignorance highlights another important question for future studies: is there a pivotal phosphoprotein, or a group of phosphoproteins, through which all kinase-activating external stimuli converge to influence the secretory process? The lack of a definitive answer to this question at present is probably due to limitations in current experimental approaches. Attempts to detect important substrates by measuring changes in phosphorylation of endogenous proteins may detect transduction elements in which the mass of phosphoprotein increases (Figure 5, upper panel) but will not necessarily detect changes in the flux of phosphate through the substrate, which may be equally important (Figure 5, lower panel). More immediately useful information can be gained by adopting the 'candidate protein' approach in which the phosphorylation of substrates known to be involved in transduction cascades is measured, although this approach is inherently unlikely to identify novel pathways or mechanisms. The rapid recent advances in cell and molecular biology are presenting ever more candidate proteins for this approach, and our ultimate understanding of the role of protein phosphorylation in the regulation of insulin secretion is probably dependent upon the application to β-cells of conceptual advances made in more experimentally accessible tissues. As the sequencing of the human genome proceeds ever more rapidly, current estimates suggest that it may contain coding sequences for more than 1000 protein kinases. The 361 studies referred to in this review have produced only limited insight into the function of a few of these enzymes in pancreatic β-cells. There is clearly much work to be done.