Phosphorylation-mimicking Mutations Research Articles

Phosphorylation of the plasma membrane H+-ATPase AHA2 by BAK1 is required for ABA-induced stomatal closure in Arabidopsis.

Stomatal opening is largely promoted by light-activated plasma membrane-localized proton ATPases (PM H+-ATPases), while their closure is mainly modulated by abscisic acid (ABA) signaling during drought stress. It is unknown whether PM H+-ATPases participate in ABA-induced stomatal closure. We established that BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) interacts with, phosphorylates and activates the major PM Arabidopsis H+-ATPase isoform 2 (AHA2). Detached leaves from aha2-6 single mutant Arabidopsis thaliana plants lost as much water as bak1-4 single and aha2-6 bak1-4 double mutants, with all three mutants losing more water than the wild-type (Columbia-0 [Col-0]). In agreement with these observations, aha2-6, bak1-4, and aha2-6 bak1-4 mutants were less sensitive to ABA-induced stomatal closure than Col-0, whereas the aha2-6 mutation did not affect ABA-inhibited stomatal opening under light conditions. ABA-activated BAK1 phosphorylated AHA2 at Ser-944 in its C-terminus and activated AHA2, leading to rapid H+ efflux, cytoplasmic alkalinization, and reactive oxygen species (ROS) accumulation, to initiate ABA signal transduction and stomatal closure. The phosphorylation-mimicking mutation AHA2S944D driven by its own promoter could largely compensate for the defective phenotypes of water loss, cytoplasmic alkalinization, and ROS accumulation in both aha2-6 and bak1-4 mutants. Our results uncover a crucial role of AHA2 in cytoplasmic alkalinization and ABA-induced stomatal closure during the plant's response to drought stress.

Ezrin interacts with L-periaxin by the “head to head and tail to tail” mode and influences the location of L-periaxin in Schwann cell RSC96

In the peripheral nervous system (PNS), Schwann cells (SCs) are required for the myelination of axons. Periaxin (PRX), one of the myelination proteins expressed in SCs, is critical for the normal development and maintenance of PNS. As a member of the ERM (ezrin-radxin-moesin) protein family, ezrin holds our attention since their link to the formation of the nodes of Ranvier. Furthermore, PRX and ezrin are co-expressed in cytoskeletal complexes with periplakin and desmoyokin in lens fiber cells. In the present study, we observed that L-periaxin and ezrin interacted in a “head to head and tail to tail” mode in SC RSC96 through NLS3 region of L-periaxin with F3 subdomain of ezrin interaction, and the region of L-periaxin (residues 1368–1461) with ezrin (residues 475–557) interaction. A phosphorylation-mimicking mutation of ezrin resulted in L-periaxin accumulation on SC RSC96 membrane. Ezrin could inhibit the self-association of L-periaxin, and ezrin overexpression in sciatic nerve injury rats could facilitate the repair of impaired myelin sheath. Therefore, the interaction between L-periaxin and ezrin may adopt a close form to complete protein accumulation and to participate in myelin sheath maintenance.

MAPK modulation of yeast pheromone signaling output and the role of phosphorylation sites in the scaffold protein Ste5.

Mitogen-activated protein kinases (MAPKs) mediate numerous eukaryotic signaling responses. They also can modulate their own signaling output via positive or negative feedback loops. In the yeast pheromone response pathway, the MAPK Fus3 triggers negative feedback that dampens its own activity. One target of this feedback is Ste5, a scaffold protein that promotes Fus3 activation. Binding of Fus3 to a docking motif (D motif) in Ste5 causes signal dampening, which was proposed to involve a central cluster of phosphorylation sites in Ste5. Here, we reanalyzed the role of these central sites. Contrary to prior claims, phosphorylation-mimicking mutations at these sites did not impair signaling. Also, the hyperactive signaling previously observed when these sites were mutated to nonphosphorylatable residues arose from their replacement with valine residues and was not observed with other substitutes. Instead, a cluster of N-terminal sites in Ste5, not the central sites, is required for the rapid dampening of initial responses. Further results suggest that the role of the Fus3 D motif is most simply explained by a tethering effect that promotes Ste5 phosphorylation, rather than an allosteric effect proposed to regulate Fus3 activity. These findings substantially revise our understanding of how MAPK feedback attenuates scaffold-mediated signaling in this model pathway.

PKG1-modified TSC2 regulates mTORC1 activity to counter adverse cardiac stress

SUMMARYThe mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis, and autophagy1. Its hyper-activation contributes to disease in many organs including the heart1,2, though broad mTORC1 inhibition risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 by modulating Rheb (Ras homolog enriched in brain). TSC2 constitutively inhibits mTORC1, but this activity is modified by phosphorylation from multiple signaling kinases to in turn inhibit (AMPK, GSK3β) or stimulate (Akt, ERK, RSK-1) mTORC1 activity3–9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here, we reveal phosphorylation or gain-or-loss of function mutations at either of two adjacent serines in TSC2 (S1365/1366 mouse; 1364/1365 human), with no prior known function, is sufficient to bi-directionally potently control growth-factor and hemodynamic-stress stimulated mTORC1 activity and consequent cell growth and autophagy. Basal mTORC1 activity, however, is unchanged. In heart, myocytes, and fibroblasts, phosphorylation occurs by protein kinase G (PKG), a primary effector of nitric oxide and natriuretic peptide signaling whose activation is protective against heart disease10–13. PKG suppression of hypertrophy and stimulation of autophagy in myocytes requires TSC2 phosphorylation. Homozygous knock-in (KI) mice expressing a phospho-silenced TSC2 (S1365A) mutation develop far worse heart disease and mortality from sustained pressure-overload (PO) due to hyperactive mTORC1 that cannot be rescued by PKG stimulation. Heterozygote SA-KI are rescued, and KI-mice expressing a phospho-mimetic (S1365E) mutation are protected. Neither KI model alters resting mTORC1 activity. Thus, TSC2 phosphorylation is both required and sufficient for PKG-mediated cardiac protection against pressure-overload. These newly identified serines provide a genetic tool to bi-directionally regulate the amplitude of stress-stimulated mTORC1 activity.

A model of the three-dimensional structure of human interferon responsive factor 1 and its modifications upon phosphorylation or phosphorylation-mimicking mutations

Interferon responsive factor 1 (IRF-1) is a pleiotropic transcription factor, possessing non-redundant biological activities that depend on its interaction with different protein partners and multiple post-translational modifications including phosphorylation. In particular, a 5′-SXXXSXS-3′ motif of the protein represents the target of the IκB-related kinases, TANK-binding kinase (TBK)-1 and inhibitor of nuclear factor kappa-B kinase (IKK)-ε. Here, a 3D model of human IRF-1 was determined by using multi-template comparative modeling and molecular dynamics approaches. Models obtained through either phosphorylation or aspartate mutation of residues 215, 219 and 221 were also calculated and compared to the wild type. Calculations indicated that each of these modifications mainly induces a rigidification of the protein structure and only slightly changes in electrostatics and hydrophobicity of IRF-1 surface, resulting in the impairment of the capacity of IRF-1 containing as partate mutations (S221D and S215D/S219D/S221D) to synergize with tumour necrosis factor (TNF)-α stimulation in inducing interferon (IFN) promoter-mediated reporter gene activation. Therefore, these changes are qualitatively correlated to the amount of negative charge located on the 215–221 segments of IRF-1 by phosphorylation or aspartate mutation. Hypotheses on the structural mechanism that governs the phosphorylation-related damping of IRF-1 activity were also drawn.Communicated by Ramaswamy H. Sarma

Mechanical stress-induced subcellular re-localization of N-terminally truncated tobacco Nt-4/1 protein

The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR.

Induction of endoplasmic reticulum (ER)-to-Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)-mediated, Golgi-independent unconventional cell-surface trafficking of the folding-deficient ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation-dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508-CFTR, such as induction of ER-to-Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C-terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation-mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation-inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER-retained ΔF508-CFTR and mediates the ER stress-induced unconventional secretion pathway.

Protein kinase C enhances human sodium channel hNav1.7 resurgent currents via a serine residue in the domain III–IV linker

Resurgent sodium currents likely play a role in modulating neuronal excitability. Here we studied whether protein kinase C (PKC) activation can increase resurgent currents produced by the human sodium channel hNav1.7. We found that a PKC agonist significantly enhanced hNav1.7-mediated resurgent currents and this was prevented by PKC antagonists. The enhancing effects were replicated by two phosphorylation-mimicking mutations and were prevented by a phosphorylation-deficient mutation at a conserved PKC phosphorylation site (Serine 1479). Our results suggest that PKC can increase sodium resurgent currents through phosphorylation of a conserved Serine residue located in the domain III–IV linker of sodium channels.

Role of Fas-Associated Death Domain-containing Protein (FADD) Phosphorylation in Regulating Glucose Homeostasis: from Proteomic Discovery to Physiological Validation

Fas-associated death domain-containing protein (FADD), a classical apoptotic signaling adaptor, participates in different nonapoptotic processes regulated by its phosphorylation. However, the influence of FADD on metabolism, especially glucose homeostasis, has not been evaluated to date. Here, using both two-dimensional electrophoresis and liquid chromatography linked to tandem mass spectrometry (LC/MS/MS), we found that glycogen synthesis, glycolysis, and gluconeogenesis were dysregulated because of FADD phosphorylation, both in MEFs and liver tissue of the mice bearing phosphorylation-mimicking mutation form of FADD (FADD-D). Further physiological studies showed that FADD-D mice exhibited lower blood glucose, enhanced glucose tolerance, and increased liver glycogen content without alterations in insulin sensitivity. Moreover, investigations on the molecular mechanisms revealed that, under basal conditions, FADD-D mice had elevated phosphorylation of Akt with alterations in its downstream signaling, leading to increased glycogen synthesis and decreased gluconeogenesis. Thus, we uncover a novel role of FADD in the regulation of glucose homeostasis by proteomic discovery and physiological validation.

The C-Terminal V5 Domain of Protein Kinase Cα Is Intrinsically Disordered, with Propensity to Associate with a Membrane Mimetic

The C-terminal V5 domain is one of the most variable domains in Protein Kinase C isoforms (PKCs). V5 confers isoform specificity on its parent enzyme through interactions with isoform-specific adaptor proteins and possibly through specific intra-molecular interactions with other PKC domains. The structural information about V5 domains in solution is sparse. The objective of this work was to determine the conformational preferences of the V5 domain from the α isoform of PKC (V5α) and evaluate its ability to associate with membrane mimetics. We show that V5α and its phosphorylation-mimicking variant, dmV5α, are intrinsically disordered protein domains. Phosphorylation-mimicking mutations do not alter the overall conformation of the polypeptide backbone, as evidenced by the local nature of chemical shift perturbations and the secondary structure propensity scores. However, the population of the “cis-trans” conformer of the Thr638-Pro639-Pro640 turn motif, which has been implicated in the down-regulation of PKCα via peptidyl-prolyl isomerase Pin1, increases in dmV5α, along with the conformational flexibility of the region between the turn and hydrophobic motifs. Both wild type and dmV5α associate with micelles made of a zwitterionic detergent, n-dodecylphosphocholine. Upon micelle binding, V5α acquires a higher propensity to form helical structures at the conserved “NFD” motif and the entire C-terminal third of the domain. The ability of V5α to partition into the hydrophobic micellar environment suggests that it may serve as a membrane anchor during the PKC maturation process.

Transient Oligomerization of the SARS-CoV N Protein – Implication for Virus Ribonucleoprotein Packaging

The nucleocapsid (N) phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genome into a helical ribonucleocapsid and plays a fundamental role during viral self-assembly. The N protein consists of two structural domains interspersed between intrinsically disordered regions and dimerizes through the C-terminal structural domain (CTD). A key activity of the protein is the ability to oligomerize during capsid formation by utilizing the dimer as a building block, but the structural and mechanistic bases of this activity are not well understood. By disulfide trapping technique we measured the amount of transient oligomers of N protein mutants with strategically located cysteine residues and showed that CTD acts as a primary transient oligomerization domain in solution. The data is consistent with the helical oligomer packing model of N protein observed in crystal. A systematic study of the oligomerization behavior revealed that altering the intermolecular electrostatic repulsion through changes in solution salt concentration or phosphorylation-mimicking mutations affects oligomerization propensity. We propose a biophysical mechanism where electrostatic repulsion acts as a switch to regulate N protein oligomerization.

Intracellular Trafficking of FXYD1 (Phospholemman) and FXYD7 Proteins in Xenopus Oocytes and Mammalian Cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.

Protein Kinase A (PKA) Phosphorylation of Na+/K+-ATPase Opens Intracellular C-terminal Water Pathway Leading to Third Na+-binding site in Molecular Dynamics Simulations

Phosphorylation is one of the major mechanisms for posttranscriptional modification of proteins. The addition of a compact, negatively charged moiety to a protein can significantly change its function and localization by affecting its structure and interaction network. We have used all-atom Molecular Dynamics simulations to investigate the structural consequences of phosphorylating the Na(+)/K(+)-ATPase (NKA) residue Ser(936), which is the best characterized phosphorylation site in NKA, targeted in vivo by protein kinase A (PKA). The Molecular Dynamics simulations suggest that Ser(936) phosphorylation opens a C-terminal hydrated pathway leading to Asp(926), a transmembrane residue proposed to form part of the third sodium ion-binding site. Simulations of a S936E mutant form, for which only subtle effects are observed when expressed in Xenopus oocytes and studied with electrophysiology, does not mimic the effects of Ser(936) phosphorylation. The results establish a structural association of Ser(936) with the C terminus of NKA and indicate that phosphorylation of Ser(936) can modulate pumping activity by changing the accessibility to the ion-binding site.

CDK-Dependent Potentiation of MPS1 Kinase Activity Is Essential to the Mitotic Checkpoint

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.

Effect of mutations imitating the phosphorylation by TRPM7 kinase on the function of the N-terminal domain of tropomodulin

It has been shown that tropomodulin 1 is phosphorylated at serine and threonine residues by TRPM7 kinase. The phosphorylation sites for TRPM7 kinase in the N-terminal functional domain of tropomodulin 1 have been identified, which include tropomyosin-binding and actin-capping regions. It has been found that the phosphorylation-mimicking mutation T54E resulted in the loss of the capping ability of the N-terminal tropomodulin domain; however, its tropomyosin-binding ability did not change. We further hypothesize that the phosphorylation of tropomodulin by TRPM7 kinase may play a role in the regulation of the dynamics of actin filaments.

Effect of Phosphorylation on αB-crystallin: Differences in Stability, Subunit Exchange and Chaperone Activity of Homo and Mixed Oligomers of αB-Crystallin and its Phosphorylation-mimicking Mutant

Phosphorylation appears to be one of the modulators of chaperone functions of small heat shock proteins. However, the role of phosphorylation is not completely understood. We have investigated the structural and functional consequences of a phosphorylation-mimicking mutation in αB-crystallin, a small heat shock protein with chaperone activity. We have used a phosphorylation-mimicking mutant, 3DαB-crystallin, in which all the three phosphorylatable serine residues are replaced with aspartic acid. 3DαB-Crystallin showed enhanced chaperone-like activity towards DTT-induced aggregation of insulin, heat-induced aggregation of citrate synthase and SDS-induced amyloid fibril formation of α-synuclein. Fluorescence and circular dichroism spectroscopic studies showed that 3DαB-crystallin exhibits lower stability towards urea-induced denaturation compared to αB-crystallin. Subunit exchange studies using fluorescence resonance energy transfer showed that 3DαB-crystallin exhibits an observable increase in subunit exchange compared to αB-crystallin. Since only part of αB-crystallin is phosphorylated in vivo, our subunit exchange studies indicate that formation of mixed oligomers between the unphosphorylated and phosphorylated subunits are likely to play a role in vivo. Our study shows that mixed-oligomer formation modulates the chaperone-like activity. We propose that the degree of phosphorylation of the αB-crystallin oligomers and temperature are key modulators to achieve a wide range of chaperone capabilities of the small heat shock protein, αB-crystallin.

Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo‐phosphorylated states

Human phospholamban (PLN), a 30 kDa homopentamer in the sarcoplasmic reticulum (SR) membrane, controls the magnitude of heart muscle contraction and relaxation by regulating the calcium pumping activity of the SR Ca(2+)-ATPase (SERCA). When PLN is not phosphorylated, it binds and inhibits SERCA. Phosphorylation of PLN at S16 or T17 releases such inhibitory effect. It remains a matter of debate whether phosphorylation perturbs the structure of PLN, which in turn affects its interaction with SERCA. Here we examine by NMR spectroscopy the structure and dynamics of PLN pentamer with a physiologically relevant, phosphorylation-mimicking mutation, S16E. Based on extensive NMR data, including NOEs, dipolar couplings, and solvent exchange of backbone amides, we conclude that the phosphorylation-mimicking mutation does not perturb the pentamer structure. However, (15)N R(1) and R(2) relaxation rates and (15)N((1)H) NOEs suggest subtle differences in the dynamics of the extramembrane portion of the protein.

Selection of Protein Phosphatase 2A Regulatory Subunits Is Mediated by the C Terminus of the Catalytic Subunit

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases all composed of a catalytic C, a structural A, and a regulatory B subunit. Assembly of the complex with the appropriate B subunit forms the key to the functional specificity and regulation of PP2A. Emerging evidence suggests a crucial role for methylation and phosphorylation of the PP2A C subunit in this process. In this study, we show that PP2A C subunit methylation was not absolutely required for binding the PR61/B' and PR72/B'' subunit families, whereas binding of the PR55/B subunit family was determined by methylation and the nature of the C-terminal amino acid side chain. Moreover mutation of the phosphorylatable Tyr(307) or Thr(304) residues differentially affected binding of distinct B subunit family members. Down-regulation of the PP2A methyltransferase LCMT1 by RNA interference gradually reduced the cellular amount of methylated C subunit and induced a dynamic redistribution of the remaining methylated PP2A(C) between different PP2A trimers consistent with their methylation requirements. Persistent knockdown of LCMT1 eventually resulted in specific degradation of the PR55/B subunit and apoptotic cell death. Together these results establish a crucial foundation for understanding PP2A regulatory subunit selection.

Phosphorylation of Both Nucleoplasmin Domains Is Required for Activation of Its Chromatin Decondensation Activity

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified a number of phosphorylated residues by mass spectrometry and generated mutants in which these amino acids are replaced by Asp to mimic the effect of phosphorylation. Our results show that, among the eight phosphoryl groups experimentally detected, four are located at the flexible N terminus, and the rest are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin.

Platelet-Derived Growth Factor Receptor β–Mediated Phosphorylation of MUC1 Enhances Invasiveness in Pancreatic Adenocarcinoma Cells

MUC1 is a heterodimeric transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in ductal adenocarcinomas. Differential phosphorylation of the MUC1 cytoplasmic tail (MUC1CT) has been associated with signaling events that influence the proliferation and metastasis of cancer cells. We identified a novel tyrosine phosphorylation site (HGRYVPP) in the MUC1CT by mass spectrometric analysis of MUC1 from human pancreatic adenocarcinoma cell lines. Analyses in vitro and in vivo showed that platelet-derived growth factor receptor beta (PDGFRbeta) catalyzed phosphorylation of this site and of tyrosine in the RDTYHPM site. Stimulation of S2-013.MUC1F cells with PDGF-BB increased nuclear colocalization of MUC1CT and beta-catenin. PDGF-BB stimulation had no significant effect on cell proliferation rate; however, it enhanced invasion in vitro through Matrigel and in vivo tumor growth and metastases. Invasive properties of the cells were significantly altered on expression of phosphorylation-abrogating or phosphorylation-mimicking mutations at these sites. We propose that interactions of MUC1 and PDGFRbeta induce signal transduction events that influence the metastatic properties of pancreatic adenocarcinoma.