The tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), is a key enzyme involved in the regulation of several biological processes including cell growth, proliferation, and death. Alterations in PTEN are tightly associated to a number of human diseases including somatic cancer (breast, endometrium, prostate, thyroid and brain cancer), tumour-syndromes (PTEN hamartoma syndromes), neurological defects (autism spectrum disorder) and altered metabolic states. Functionally, PTEN is a phosphatase with activity towards lipid and protein substrates. The lipid phosphatase activity of PTEN has been well characterized and is undoubtedly linked to dephosphorylation of the second messenger phosphatidyl-inositol-3,4,5 trisphosphate (PIP3) to phosphatidyl-inositol-4,5 bisphosphate (PIP2), and is commonly associated with inhibition of the pro-survival PI3K/AKT signal transduction pathway. In contrast, the phosphatase activity of PTEN towards protein substrates remains poorly understood and the few PTEN phospho-targets identified so far contentious. Here, using unique Pten mutant knock-in mice, we investigate the phosphorylation signatures driven by loss of PTEN catalytic function and explore the status of PTEN-regulated pathways beyond the PI3K pathway. These studies provide key insight into mechanisms of PTEN-dependent tumour suppression and have important therapeutic implications for a range of clinical conditions.