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Interaction of prolactin (PRL) with its receptor (PRLR) leads to activation of Jak and Src family tyrosine kinases. The PRL} growth hormone}cytokine receptor family conserves a proline-rich sequence in the cytoplasmic juxtamembrane region (Box 1) required for association and subsequent activation of Jaks. In the present work, we studied the mechanisms underlying c-Src kinase activation by PRL and the role that Jak2 plays in this process. PRL addition to chicken embryo fibroblasts (CEF) expressing the rat PRLR long form resulted in activation of c-Src and Jak2 and in tyrosine phosphorylation of the receptor. Receptor phosphorylation was due to associated Jak2, since in cells expressing either a Box 1 mutated PRLR (PRLR %P-A), which i