Protein Phosphatases in Cellular Signaling / Ferenc Erdődi

Personal data: 

Ferenc Erdődi, D. Sc.
E-mail: erdodimed.unideb.hu
http://www.medchem.dote.hu/hu/kutatas/munkacsoport/myosin.htm

Research: 

This research group studies the regulatory role of protein phosphorylation and dephosphorylation in cellular processes. Our major interest is the structure, localization and regulation of serine-threonine specific (Ser/Thr) protein phosphatase type 1 (PP1) and type 2A (PP2A). The cellular functions of these enzymes are probed with cell-permeable phosphatase inhibitory toxins (okadaic acid, calyculin A, tautomycin, etc.). Type and subunit specific antibodies are applied for localization of these proteins in subcellular fractions by Western blots or by florescent microscopy. We express and purify recombinant catalytic and regulatory subunits of phosphatases, and generate truncated as well as site-directed mutants to identify the regions and residues involved in the interactions and regulation. Phosphatase activity may be controlled by phosphorylation of the regulatory and inhibitory proteins; therefore one of our major interests is to identify protein kinases that catalyze phosphorylation of the phosphatase-associating proteins and to dissect the signaling pathways involved in these regulatory processes. The functions of PP1 and PP2A enzymes in the physiological responses upon stimuli of neuronal cells, cancer cells, and endothelial cells have been investigated.

Recently, our studies have focused on the role of protein-protein interactions in the mediation of activity of protein phosphatases including association of the catalytic subunits with regulatory (targeting) subunits and inhibitory proteins. The methods applied include measurements of protein-protein interactions in real time with surface plasmon resonance technique (Biacore), immunological methods (FarWestern analysis, confocal immunfluorescence) and other biochemical techniques (pull-down assays based on affinity chromatography, gel-filtration, etc.). We have localized the PP1 type myosin phosphatase catalytic (PP1c) and targeting (MYPT) subunits in different subcellular compartments (cytoplasm, nucleus, nucleolus) of various neuronal cells. PP1c, Rho-kinase and synaptophysin were identified as major proteins interacting with MYPT in cerebral cortex. In HepG2 cells it was shown that phosphorylation of MYPT by Rho-kinase resulted in inhibition of myosin phosphatase activity which correlated with increased stress fiber formation and decreased cell migration.
Several types of protein phosphatases are intimately involved in the regulation of endothelial barrier function. TGF-β-inhibited membrane-associated protein (TIMAP) is regarded as a member of the MYPT family, and it is expressed at high levels in endothelial cells (EC). TIMAP binds preferentially the β (also termed as ) isoform of the catalytic subunit of PP1 from pulmonary artery EC suggesting that TIMAP is a novel regulatory subunit of PP1 in EC. TIMAP depletion by siRNA technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP), and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed co-localization of TIMAP with membrane/cytoskeletal protein, moesin.

Currently we study the role of PP1 and PP2A in the regulation of the phosphorylation level of proteins in leukemic and other cancer cells. We investigate the influence of protein phosphatase activation/inhibition on cell death induced by chemotherapeutic agents. Initial results indicate that the myosin phosphatase is involved in the dephosphorylation of retinoblastoma protein; therefore it can mediate G1/S transition in cell cycle. Inhibition of protein phosphatase activity results in increased cell survival when cell death is induced by daunorubicin implicating protein phosphatases in the chemoresistance of cancer cells.
Myosin phosphatase and Rho-kinase are also involved in the regulation of neurotransmitter release from isolated synaptosomes and neuronal cells. Inhibition of phosphatase activity suppresses neurotransmitter release induced by depolarization, whereas inhibition of Rho-kinase increases neurotransmitter release.
The role of TIMAP in different physiological functions of endothelial cells is further investigated focusing on the induction of phosphorylation processes. Activation of cAMP/protein kinase A (PKA) cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On contrary, in TIMAP-depleted EC forskolin failed to affect the level of moesin phosphorylation at the cell edges. The goal of our current research is to clarify the role of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

Photos: 

 

 

Figure 1.

Interaction of the catalytic subunit (PP1c) with myosin phosphatase target subunit-1 (MYPT1) in the myosin phosphatase holoenzyme and the regulation of the phosphatase activity by interaction of PP1c with inhibitory toxins and proteins (CPI-17-P) as well as via phosphorylation of the MYPT1 subunit by Rho-kinase (ROK).

 

 

 

 

Figure 2.

Localization of MYPT1 (green) and retinoblastoma protein (pRb, red) in THP-1 leukemic cells. Colocalization of MYPT1 and pRb in control and daunorubicin (DNR) treated cells in the nucleus (D, H). Translocation of MYPT1 from the nucleus into the cytoplasm upon phosphatase inhibitory toxin (calyculin-A) treatment of the cells (I, M).

 

 

 

 

Figure 3.

Immunofluorescent staining of TIMAP in endothelial cells.

 

 

 

 

 

Publications: 

Representative publications:

Wooldridge AA, MacDonald JA, Erdodi F, Ma CY, Borman MA, Hartshorne DJ, Haystead TAJ
Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of serine 695 in response to cyclic nucleotides
J BIOL CHEM 279:(33) 34496-34504 (2004)

Lontay B, Serfozo Z, Gergely P, Ito M, Hartshorne DJ, Erdodi F
Localization of myosin phosphatase target subunit 1 in rat brain and in primary cultures of neuronal cells
J COMP NEUROL 478:(1) 72-87 (2004)

Hartshorne DJ, Ito M, Erdodi F
Role of protein phosphatase type 1 in contractile functions: Myosin phosphatase
J BIOL CHEM 279:(36) 37211-37214 (2004)

Tar K, Csortos C, Czikora I, Olah G, Ma SF, Wadgaonkar R, Gergely P, Garcia Jgn, Verin AD
Role of Protein Phosphatase 2A in The Regulation of Endothelial Cell Cytoskeleton Structure
J CELL BIOCHEM 98:(4) 931-953 (2006)

Csortos C, Kolosova I, Verin AD
Regulation of vascular endothelial cell barrier function and cytoskeleton structure by protein phosphatases of the PPP family
AM J PHYSIOL-LUNG C 293:(4) L843-L854 (2007)

Recent publications:

Muranyi A, Derkach D, Erdodi F, Kiss A, Ito M, Hartshorne DJ
Phosphorylation of Thr695 and Thr850 on the myosin phosphatase target subunit: Inhibitory effects and occurrence in A7r5 cells
FEBS LETT 579:(29) 6611-6615 (2005)

Lontay B, Kiss A, Gergely P, Hartshorne DJ, Erdodi F
Okadaic Acid Induces Phosphorylation And Translocation of Myosin Phosphatase Target Subunit 1 Influencing Myosin Phosphorylation, Stress Fiber Assembly And Cell Migration in Hepg2 Cells
CELL SIGNAL 17:(10) 1265-1275 (2005)

Wu Y, Muranyi A, Erdodi F, Hartshorne DJ
Localization of myosin phosphatase target subunit and its mutants
J MUSCLE RES CELL M 26:(2-3) 123-134 (2005)

Kiss A, Lontay B, Bécsi B, Márkász L, Oláh É, Gergely P, Erdődi F
Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A
CELL SIGNAL 20: 2059-2070 (2008)

Csortos C, Czikora I, Bogatcheva NV, Adyshev DM, Poirier C, Olah G, Verin AD
TIMAP is a positive regulator of pulmonary endothelial barrier function
AM J PHYSIOL-LUNG C 295:(3) L440-L450 (2008)