Diacylglycerol (DAG) kinases (Dgk), which phosphorylate DAG to generate phosphatidic acid, act as either positive or negative key regulators of cell signaling. We previously showed that Src mediates growth factors-induced activation of Dgk-alpha, whose activity is required for cell motility, proliferation and angiogenesis. Here, we demonstrate that both hepatocytes growth factor (HGF) stimulation and v-Src transformation induce tyrosine phosphorylation of Dgk-alpha on Y335, through a mechanism requiring its proline-rich C-terminal sequence. Moreover, we show that both proline-rich sequence and phosphorylation of Y335 of Dgk-alpha mediate: (i) its enzymatic activation, (ii) its ability to interact respectively with SH3 and SH2 domains of Src, (iii) its recruitment to the membrane. In addition, we show that phosphorylation of Dgk-alpha on Y335 is required for HGF-induced motility, while its constitutive recruitment at the membrane by myristylation is sufficient to trigger spontaneous motility in absence of HGF. Providing the first evidence that tyrosine phosphorylation of Dgk-alpha is required for growth-factors-induced activation and membrane recruitment, these findings underscore its relevance as a rheostat, whose activation is a threshold to elicit growth factors-induced migratory signaling.