Phosphorylation of threonine 61 by cyclin a/Cdk1 triggers degradation of stem-loop binding protein at the end of S phase.

Progression through the cell cycle is driven by a class of protein kinases, the cyclin-dependent kinases (cdk's), which are sequentially activated as cells progress from one cell cycle stage to the next. In particular, progress into S phase requires the activation of cyclin E/Cdk2, ultimately resulting in the initiation of DNA replication, and progression through mitosis requires the activation of cyclin B/Cdk1 (cdc2), resulting in nuclear envelope breakdown and chromosome condensation (27). Cyclin A/Cdk2 activity is required for continued progression through S phase (36). The targets of the cdk's are activated or inactivated by phosphorylation. A second critical regulatory mechanism for cell cycle progression is targeted proteolysis of key protein regulators (13). These include the cyclin subunits of the cdk's, critical proteins in initiation of DNA replication, and proteins responsible for maintaining chromosome pairing (6). While much is known about the events that must occur for cells to transit from G1 to S phase and for cells to enter and exit mitosis, much less is known about the molecular events at the end of S phase, as cells progress from S to G2 phase. S phase is characterized by replication of the chromosomes, and at the same time DNA is replicated, histone proteins must be synthesized to provide histones to assemble the newly replicated chromatin. Histone mRNAs are cell cycle regulated, and their expression is restricted to S phase. The metazoan replication-dependent histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated. Instead, they end in a conserved stem-loop sequence (18). Since the replication-dependent histone genes lack introns, the only processing reaction required for histone mRNA biosynthesis is cleavage of the nascent transcript to form the 3′ end of the mRNA (18). The 3′ end of histone mRNAs is a major cis element responsible for the coordinated regulation of histone mRNA levels (17). The 3′ end is bound by the stem-loop binding protein (SLBP), which is required for both histone pre-mRNA processing and histone mRNA translation (18). The processing of histone pre-mRNA is cell cycle regulated, as is the stability of histone mRNAs (11). SLBP is also a cell cycle-regulated protein, accumulating only in S-phase cells (34), and regulation of SLBP levels is an important component of the cell cycle regulation of histone mRNA. SLBP is rapidly degraded at the end of S phase as a result of phosphorylation of two threonines in an SFTTP sequence (amino acids 58 to 62). Proline 62 is also required for SLBP degradation (37), as is a KRKL sequence (amino acids 95 to 98) that is a consensus cyclin binding site (1-3, 5, 30) thought to primarily interact with the S-phase cyclins A and E (16). We report here that cyclin A/Cdk1 initiates SLBP degradation at the end of S phase by phosphorylating Thr61. Subsequent to that phosphorylation, CK2 phosphorylates T60, and the doubly phosphorylated SLBP is targeted for degradation. Cyclin A/Cdk1 activity is cell cycle regulated and is the major activity in late-S-phase cells that phosphorylates Thr61 of SLBP. We propose that cyclin A/Cdk1 is activated near the end of S phase, resulting in the degradation of SLBP and downregulating histone mRNA synthesis. It is likely that cyclin A/Cdk1 phosphorylates other targets at the same time, during the transition from S phase to G2 phase.