The PI3-kinase/Akt pathway regulates many biological processes including insulin-regulated GLUT4 insertion into the plasma membrane (PM). However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this we examined insulin regulation of components within the signalling cascade in a quantitative kinetic and dose response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the PM strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with pThr642 grouped with other Akt substrates. Further experiments suggested that aPKCzeta phosphorylates AS160 at Ser588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signalling components for biological specificity.