Nucleic Acids Research, 2010, Vol. 38, No. 19 6617 Figure 5. Phosphorylation at S300 or S302 locates Drosha to the nucleus. Experimental procedures were the same as in Figure 1B but used different constructs as indicated. (A) Expression of GFP–Drosha with Serine300 mutated to Alanine showing nuclear localization. (B) Expression of GFP– Drosha with Serine302 mutated to Alanine also showed nuclear localization. (C) Expression of GFP–Drosha with both serines mutated to alanines showing diffuse cellular localization similar to that of GFP without NLS. (D) Expression of GFP–Drosha with Serine300 mutated to glutamic acid showed nuclear localization. (E) Expression of GFP–Drosha with Serine302 mutated to aspartic acid showing nuclear localization. (F) Expression of GFP–Drosha with Serine300 mutated to glutamic acid and Serine302 mutated to aspartic acid also showed nuclear localization. and phosphatases (dephosphorylation) are involved. Phosphorylation plays critical roles in signal transduction, protein localization and degradation (33–35). We have consistently identiﬁed Drosha phosphorylation at S300 and S302 from immunoprecipitated endogenous and overexpressed Drosha by mass spectrometry analysis. The potential kinases which phosphorylate S300 and S302 could include cyclin dependent kinase (CDK)5, glycogen synthase kinase(GSK)3, mitogen activated protein kinase(MAPK)3, MAPK2, MAPK8, protein kinase A(PKA), protein kinase Ca(PKCa), protein kinase B(PKB), casein kinaseI (CKI), etc. All of our data indicate that these serine sites might be constitu- tively phosphorylated by multiple kinases. Identiﬁcation of the speciﬁc enzymes which affect Drosha phosphoryl- ation status may reveal new insights into miRNA