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Abstract: Reversible protein phosphorylation is one of the most dynamic post-translational modifications (PTMs) regulating eukaryotic cell functions. It is catalyzed by two opposing protein families, protein kinases (PKs) and phosphatases (PPs). PKs represent enzymes with high substrate specificity that catalyze reversible protein phosphorylation events through the transfer of the γ-phosphoryl group from adenosine triphosphate (ATP) to the target substrate. Such enzymes play an important role in the regulation of a wide range of plant cellular and developmental processes such as plant metabolism, stress tolerance, and plant flowering. Despite the transcriptional and genetic networks of flowering being well-established, the impact of post-translational modifications such as reversible protein phosphorylation remains largely unknown. Here, PROTEIN KINASE 4 (PXK4), a novel flowering-related protein in Arabidopsis thaliana (Arabidopsis) is described. Initial phosphoproteomic screening of pxk4 T-DNA insertional mutant line 2 (pxk4-2) found significant decreases in the phosphorylation of multiple key flowering transition proteins. Further, phenotypic characterization of multiple pxk4 mutant alleles (pxk4-2, -4, -5) revealed an early flowering phenotype across all alleles, which was supported by qPCR analysis of known flowering genes in pxk4-2 plants that found significantly reduced FLC and elevated FT expression. Subsequent protein-protein interactome analysis of the putative PXK4 substrate and flowering transition regulator HISTONE MONO-UBIQUITINATION 2 (HUB2), using tandem affinity purification fusion proteins expressed in WT (Col-0) and pxk4-2 found that loss of PXK4 impacted various flowering regulatory complexes, indicating a potential mechanism by which PXK4 regulates flowering transition. Taken together, these findings suggest that PXK4 plays a key role in flowering through the likely regulation of multiple proteins involved flowering transition. In particular, results suggest PXK4 functions to regulate HUB2 complex formation in response to photoperiod, ultimately resulting in the modulation of FLC and FT levels.Reversible protein phosphorylation is one of the most dynamic post-translational modifications (PTMs) regulating eukaryotic cell functions. It is catalyzed by two opposing protein families, protein kinases (PKs) and phosphatases (PPs). PKs represent enzymes with high substrate specificity that catalyze reversible protein phosphorylation events through the transfer of the γ-phosphoryl group from adenosine triphosphate (ATP) to the target substrate. Such enzymes play an important role in the regulation of a wide range of plant cellular and developmental processes such as plant metabolism, stress tolerance, and plant flowering. Despite the transcriptional and genetic networks of flowering being well-established, the impact of post-translational modifications such as reversible protein phosphorylation remains largely unknown. Here, PROTEIN KINASE 4 (PXK4), a novel flowering-related protein in Arabidopsis thaliana (Arabidopsis) is described. Initial phosphoproteomic screening of pxk4 T-DNA insertional mutant line 2 (pxk4-2) found significant decreases in the phosphorylation of multiple key flowering transition proteins. Further, phenotypic characterization of multiple pxk4 mutant alleles (pxk4-2, -4, -5) revealed an early flowering phenotype across all alleles, which was supported by qPCR analysis of known flowering genes in pxk4-2 plants that found significantly reduced FLC and elevated FT expression. Subsequent protein-protein interactome analysis of the putative PXK4 substrate and flowering transition regulator HISTONE MONO-UBIQUITINATION 2 (HUB2), using tandem affinity purification fusion proteins expressed in WT (Col-0) and pxk4-2 found that loss of PXK4 impacted various flowering regulatory complexes, indicating a potential mechanism by which PXK4 regulates flowering transition. Taken together, these findings suggest that PXK4 plays a key role in flowering through the likely regulation of multiple proteins involved flowering transition. In particular, results suggest PXK4 functions to regulate HUB2 complex formation in response to photoperiod, ultimately resulting in the modulation of FLC and FT levels.