Dual kinase-mediated regulation of PITK by CaMKII and GSK3

doi:10.1016/j.cellsig.2006.08.009

Cellular Signalling

Volume 19, Issue 3, March 2007, Pages 593-599

https://doi.org/10.1016/j.cellsig.2006.08.009 Get rights and content

Abstract

Phosphatase Interactor Targeting K protein (PITK) was previously identified as a novel PP1 targeting subunit implicated in modulating the phosphorylation of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) [Kwiek NC, Thacker DF, Datto MB, Megosh HB, Haystead TA. Cell Signal 18 (10) (2006) 1769.]. Through the phosphorylation of PITK at S1013 and S1017 (residues that flank or reside within a PP1C-binding motif), the binding of the PP1 catalytic subunit to PITK, and subsequently the activity of the holoenzyme, are discretely controlled. Herein, we demonstrate that PITK phosphorylation at S1013 and S1017 also dictates the subcellular localization of the holoenzyme. Whereas both wildtype-and an S1013,1017D-PITK mutant displayed a speckled nuclear localization, a constitutively dephosphorylated form of PITK (S1013,1017A-PITK) resulted in a diffuse localization throughout the cell including the cytoplasm. Additionally, through the use of unbiased proteomics techniques, we provide evidence for a dual kinase-mediated regulation of the PITK holoenzyme whereby PITK phosphorylation at S1017 is catalyzed by calcium/calmodulin-dependent kinase II-delta (CaMKIIδ), promoting the subsequent phosphorylation of S1013 by glycogen synthase kinase-3 (GSK3) in vitro. Taken together, our findings provide further insight into the regulation of PITK, PP1, and hnRNP K by reversible phosphorylation.

Introduction

The multifaceted role of PP1 in very distinct biological processes is well-established, including functions in cell cycle regulation, glycogen metabolism, muscle contractility, morphogenesis, spermatogenesis, and memory/learning [2]. With a crucial role in such diverse functions, the cell necessitates a mechanism to impart distinct regulation upon PP1 in order to maintain physiological specificity. As such, the ability of PP1 activity to be modulated independently and completely within the cell results from the mutually exclusive interaction of its catalytic subunit with an assortment of regulatory subunits. This complex formation then dictates PP1 function through an alteration of substrate specificity, subcellular localization, and/or subsequent regulation.

In most putative regulatory subunits, a conserved peptide sequence, namely the ‘KVxF motif’, appears critical for interaction with PP1C [3], [4]. This peptide motif contacts a hydrophobic groove on PP1C, distal from the active site. Binding through the KVxF motif does not directly induce conformation changes to PP1 but rather serves as an anchor for the initial binding of the regulatory subunits, thereby promoting the binding of secondary sites that then affect the activity and substrate specificity of PP1 [5]. The finding that multiple regulatory subunits bind PP1C through, at least in part, a short, degenerate sequence motif suggested that PP1 is subject to combinatorial control that relies on the competition of its various regulators for a limited availability of interaction sites. Hence, an arsenal of regulatory subunits may interact with PP1C in a mutually exclusive manner, generating a large variety of holoenzyme combinations with distinct affinities for substrates.

Upon formation, a given PP1C/regulatory subunit holoenzyme is subject to a variety of extra-and intracellular signals that impinge on the integrity and function of this complex through multiple mechanisms, the most prevalent of which is reversible phosphorylation. Although a small fraction of PP1C molecules may be regulated by the direct phosphorylation of the catalytic subunit [6], regulation of PP1 activity and specificity generally occurs through the phosphorylation of its regulatory subunits. The ongoing study of several targeting subunits including G_M, neurabin, and NIPP1 have demonstrated that phosphorylation of serine residues within or near the conserved (R/K)(V/I)XF motif diminishes PP1C binding and subsequently subunit-directed dephosphorylation toward a given substrate [7], [8], [9], [10], [11]. In contrast, phosphorylation of inhibitory subunits such as inhibitor-1, DARPP-32, and CPI-17 creates an additional binding site for PP1C, thereby strengthening their inhibitory properties [12], [13]. Thus, the cell has evolved sophisticated signaling networks whereby the actions of both serine/threonine kinases and phosphatases are exquisitely regulated in a reciprocal fashion; that is, phosphatases such as PP1 regulate the actions of kinase-mediated phosphorylation events whereas kinases modulate the activity of PP1-mediated dephosphorylation.

In a previous study in our laboratory [1], we utilized proteomics technologies to identify and characterize PITK (Phosphatase Interactor Targeting K-protein), a novel PP1 targeting subunit that functions to modulate the phosphorylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K or K protein). Furthermore, we unbiasedly defined the regulation of PITK by a combination of Edman degradation and MS analysis, demonstrating that PITK is reversibly phosphorylated in vivo at S1013 and S1017, residues that flank or reside within a putative KVxF motif. Incidentally, the phosphorylation of PITK at these specific residues altered PP1 binding and subsequent PITK-directed dephosphorylation of hnRNP K. Due to the importance of these residues in dictating the function of the PITK/PP1 holoenzyme, we initiated this study to further examine the implications of PITK phosphorylation at S1013 and S1017 as well as to identify the relevant S1013- and S1017-PITK kinase(s). Herein, we demonstrate that PITK phosphorylation at these residues, in addition to altering PP1 interaction, also dictates the subcellular localization of the holoenzyme. Furthermore, we provide evidence for a dual kinase-mediated regulation of a PP1 holoenzyme whereby PITK phosphorylation at S1017 is catalyzed by calcium/calmodulin-dependent kinase II-delta (CaMKIIδ), promoting the subsequent phosphorylation of S1013 by glycogen synthase kinase-3 (GSK3) in vitro. Collectively, this work provides further insight into the regulation and biology of the PITK holoenzyme

Section snippets

Materials

All chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich Co., and radiochemicals from MP Biochemicals. All solutions were prepared with Millipore MilliQ water. Cell culture lines were obtained from the Duke University Comprehensive Cancer Center Cell Culture Facility.

Antibodies

The following antibodies were utilized at the dilutions indicated in brackets: anti-FLAG M2 [1:5000 for immunoblotting, 1:200 for immunofluorescence], pan anti-PP1C [1:1000, Santa Cruz Biotechnology], goat

Results

In a quest to further characterize other putative effects of PITK phosphorylation in vivo, we examined the subcellular localization of phosphomutant (S1013,1017D; S1013,1017A)-PITK constructs by indirect immunofluorescence. FLAG constructs of the phosphomutants were transfected into HEK293T cells and visualized by fluorescence microscopy. Similar to that observed with a GFP-fusion protein [1], wildtype-PITK exhibited compartmentalization in the nucleus (Fig. 1). The S1013,1017D-PITK mutant,

Discussion

Due to its efficiency and reversibility, the inducible phosphorylation of targeting subunits represents an ideal method for regulating PP1 holoenzyme composition and function. The rapid dissociation of PP1C and its regulatory subunit by phosphorylation within the KVxF motif facilitates the exchange of targeting proteins within the holoenzyme, thereby modulating PP1 function depending on the impinging signaling network. This dissociation likely occurs through a disruption of the PP1 holoenzyme

Acknowledgements

We thank Elizabeth Herrick Snyder for help in figure preparation. This work was funded by National Institutes of Health grant RO1DK052378-09 awarded to T.A.J.H.

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Dual kinase-mediated regulation of PITK by CaMKII and GSK3

Abstract

Introduction

Section snippets

Materials

Antibodies

Results

Discussion

Acknowledgements

Cell Signal.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Mol. Cell. Proteomics

J. Biol. Chem.

Trends Biochem. Sci.

J. Biol. Chem.

Physiol. Rev.

Nature

EMBO J.

Proc. Natl. Acad. Sci. U. S. A.