Profilin 2 threonine 90 phosphorylation

Abstract

Profilins are small actin binding proteins that interact with a plethora of ligands to fulfill their functions. These interactions were shown to be regulated by phosphorylation, also in relation to pathological conditions. However, most studies focused on the predominant PFN1 isoform. I previously identified threonine 90 as a novel phosphorylation site on PFN2 in the mouse brain, however its function remains elusive. In this thesis, I show that phospho-mimetic PFN2-T90D accelerates spontaneous and polymerase catalyzed actin polymerization. While thermodynamic binding of PFN2-T90D to actin and synthetic proline-streches was unaltered <em>in vitro</em>, actin and the majority of proline-rich ligands were reduced in PFN2-T90D complexes pulled-down from mouse brain extracts. Interestingly, I show that the structure of PFN2 is severely destabilized at physiological temperature resulting in protein aggregation <em>in vitro</em>. Accordingly, <em>Pfn2^T90D</em> transfected cells and the newly generated <em>Pfn2^T90D/T90D</em> mouse model did not express detectable amounts of PFN2 likely due to protein aggregation and subsequent degradation. In phospho-deficient <em>Pfn2^T90A/T90A</em> mice, on the other hand, both brain morphology and synaptic transmission were unaltered, nevertheless, compared to the wt, PFN2 phosphorylation was significantly reduced only in the spinal cord. Consistently, steady-state actin filaments were shorter in spinal cords of <em>Pfn2^T90D/T90D</em> mice, suggesting a possible function of T90 phosphorylation in spinal cord neurons. Finally, using a mass spectrometry based kinase trapping approach and an in vitro kinase assay, I show that BRSK1 is a novel PFN2 kinase and that PKA-mediated PFN2 phosphorylation is enhanced on the phospho-mimetic T90D variant. In summary, the data presented show that T90 phosphorylation is a functional post-translational modification of PFN2 which directly influences its actin regulatory function and its interactome. Furthermore, it might also be a mechanism to regulate PFN2 turnover, weakening the structure of PFN2.