Abstract
The Drosophila homolog of cAMP-response element-binding protein (CREB), dCREB2, exists with serine 231, equivalent to mammalian serine 133, in a predominantly phosphorylated state. Thus, unlike the mammalian protein, the primary regulation of dCREB2 may occur at a different step from serine 231 phosphorylation. Although bacterially expressed dCREB2 bound cAMP-response element sites, protein from Drosophila extracts was unable to do so unless treated with phosphatase. Phosphorylation of recombinant protein by casein kinase (CK) I or II, but not calcium-calmodulin kinase II or protein kinase A, inhibited DNA binding. Up to four conserved CK sites likely to be phosphorylated in vivo were responsible for this effect, and these sites were phosphorylated by a kinase present in Drosophila cell extracts that biochemically resembles CKII. We propose that the relative importance of different signaling pathways in regulating CREB activity may differ between Drosophila and mammals. In Drosophila, the dephosphorylation of CK sites appears to be the major regulatory step, while phosphorylation of serine 231 is necessary but secondary.
Highlights
In mammals, the cAMP-response elementbinding protein (CREB) family consists of three different genes: CREB, CREM, and ATF-1 [1]
We propose that the casein kinase (CK) sites of dCREB2 may provide this mechanism by regulating DNA binding
In support of this model, we demonstrated that phosphorylation of the CK sites prevents DNA binding, while dephosphorylation allows dCREB2 to bind
Summary
The CREB family consists of three different genes: CREB, CREM, and ATF-1 [1]. Sequence alignment of CREB genes from many species identifies two regions of the protein that are highly conserved [1], a basic region-leucine zipper structural motif involved in dimerization and DNA binding and a 60-amino acid kinase-inducible domain (KID) (Fig. 1). CREB activity is regulated by phosphorylation of sites within the KID. Ser-129 is believed to be a substrate for glycogen synthase kinase 3 Phosphorylation of this site occurs only after phosphorylation of Ser-133 and can function to reduce DNA binding of CREB [15, 16]. Ser-231, which is homologous to the critical mammalian Ser133 residue, can be phosphorylated by PKA, and a serine to alanine mutation at this site completely abolishes activity [19]. The CK sites of CREB are phosphorylated during early S phase in a cell cycle-dependent manner, the role of
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