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Comparative analysis of phosphorylation of translational initiation and elongation factors by seven protein kinases
P T Tuazon1, W C Merrick, J A Traugh
1Department of Biochemistry, University of California, Riverside 92521.
Abstract:
Four initiation factors (eIF-2, -3, -4B, and -4F), previously shown to be phosphorylated in vivo, are each phosphorylated to a significant extent in vitro (greater than 0.3 mol of phosphate/mol of factor) by at least three different protein kinases. An S6 kinase from liver, an active form of protease-activated kinase II which modifies the same sites on S6 as those phosphorylated in vivo in response to mitogens, phosphorylates the beta subunit of eIF-2, eIF-3 (p120-p130), eIF-4B, and eIF-4F (p220). The Ca2+, phospholipid-dependent protein kinase phosphorylates eIF-2 beta, eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220, p25). The cAMP-dependent protein kinase significantly modifies eIF-4B and, to a lesser extent, eIF-3 (p130). Casein kinase I incorporates phosphate only into eIF-4B, but to a limited extent. Casein kinase II phosphorylates eIF-2 beta, eIF-3 (p170, p120), and eIF-4B, while protease-activated kinase I modifies eIF-3 (p170, p120-p130), eIF-4B, and eIF-4F (p220). The mitogen-stimulated S6 kinase from 3T3-L1 cells, activated in response to insulin, does not phosphorylate any of the initiation factors. There is no significant incorporation of phosphate into eIF-2 alpha or -gamma, eIF-4A, eIF-4C, eIF-4D, EF-1, or EF-2 by any of the protein kinases examined. Phosphopeptide mapping of tryptic digests of the phosphorylated subunits shows that the individual protein kinases modify different sites. The sites phosphorylated in vitro reflect those modified in vivo as shown with eIF-4F in concomitant studies with reticulocytes treated with tumor-promoting phorbol ester (Morley, S.J., and Traugh, J. A. J. Biol. Chem., in press). Thus, we have identified multipotential protein kinases which modify four initiation factors phosphorylated in vivo and have shown that phosphorylation of these translational components can be coordinately regulated.
Insights
Multiple protein kinases phosphorylate key translation initiation factors in vitro, mirroring in vivo modifications. This suggests coordinated regulation of protein synthesis through these phosphorylation events.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- Protein phosphorylation plays a crucial role in regulating cellular processes, including translation initiation.
- Four eukaryotic initiation factors (eIF-2, eIF-3, eIF-4B, and eIF-4F) are known to be phosphorylated in vivo.
- Identifying the specific kinases responsible for these modifications is essential for understanding translational control.
Purpose of the Study:
- To investigate the in vitro phosphorylation of four key translation initiation factors (eIF-2, eIF-3, eIF-4B, and eIF-4F) by various protein kinases.
- To determine if in vitro phosphorylation sites correspond to those modified in vivo.
- To explore the potential for coordinated regulation of translation initiation through kinase-mediated phosphorylation.
Main Methods:
- In vitro kinase assays using purified protein kinases (S6 kinase, protease-activated kinase II, Ca2+, phospholipid-dependent protein kinase, cAMP-dependent protein kinase, casein kinase I, casein kinase II, protease-activated kinase I) and translation initiation factors.
- Quantification of phosphate incorporation into initiation factors.
- Phosphopeptide mapping of tryptic digests of phosphorylated subunits.
- Comparison of in vitro phosphorylation sites with in vivo modified sites.
Main Results:
- Multiple protein kinases significantly phosphorylated eIF-2 (beta subunit), eIF-3, eIF-4B, and eIF-4F in vitro.
- Specific kinases exhibited distinct substrate specificities and modified different sites within the initiation factors.
- In vitro phosphorylation patterns largely reflected in vivo modifications, particularly for eIF-4F.
- Certain initiation factors (e.g., eIF-2 alpha/gamma, eIF-4A) were not significantly phosphorylated by any tested kinase.
Conclusions:
- Several multipotential protein kinases can phosphorylate key translation initiation factors in vitro.
- The identified kinases and their phosphorylation sites provide insights into the mechanisms of translational control.
- These findings suggest that phosphorylation of translation initiation factors can be coordinately regulated, impacting protein synthesis.
- The study establishes a link between specific kinases and the in vivo phosphorylation of translational components.