The p21-activated serine-threonine kinase (PAK1) is activated by small GTPase-dependent and -independent mechanisms and regulates cell motility. Both PAK1 and the hormone prolactin (PRL) have been implicated in breast cancer by numerous studies. We have previously shown that the PRL-activated tyrosine kinase JAK2 (Janus tyrosine kinase 2) phosphorylates PAK1 in vivo and identified tyrosines (Tyr) 153, 201, and 285 in the PAK1 molecule as sites of JAK2 tyrosyl phosphorylation. Here, we have used human breast cancer T47D cells stably overexpressing PAK1 wild type or PAK1 Y3F mutant in which Tyr(s) 153, 201, and 285 were mutated to phenylalanines to demonstrate that phosphorylation of these three tyrosines are required for maximal PRL-dependent ruffling. In addition, phosphorylation of these three tyrosines is required for increased migration of T47D cells in response to PRL as assessed by two independent motility assays. Finally, we show that PAK1 phosphorylates serine (Ser) 2152 of the actin-binding protein filamin A to a greater extent when PAK1 is tyrosyl phosphorylated by JAK2. Down-regulation of PAK1 or filamin A abolishes the effect of PRL on cell migration. Thus, our data presented here bring some insight into the mechanism of PRL-stimulated motility of breast cancer cells.

JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin-and interferon gamma (IFN-␥)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. JAK2 is a tyrosine kinase that is activated by two-thirds of the cytokine/hematopoietin receptor superfamily. JAK2 activation initiates a variety of downstream signaling events that lead to diverse physiological responses to cytokines, including regulation of body growth, hematopoiesis, satiety, lactation, and various immune functions. Upon activation, receptor-bound JAK2 phosphorylates specific tyrosine residues of its downstream targets, activating cell survival/proliferation-promoting signaling pathways (1). One target of JAK2 is a family of transcription factors termed signal transducers and activators of transcription (STATs). STATs exist within the cytoplasm in a latent or inactive state; they are recruited by cytokine receptor complexes through interaction between the receptor or JAK phosphotyrosines and the Src homology 2 (SH2) domain of the STAT protein. Several additional kinase cascades may be activated by JAK2, including phosphoinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Three mechanisms of JAK2 activation at the genetic level have been implicated in hematologic malignancies: point mutations, translocations, and amplifications (reviewed in reference 2). Mutations within the pseudokinase domain of JAK2 were detected in a spectrum of myeloproliferative diseases. A V617F gain-of-function point mutation is the cause of ϳ90% of all polycythemia vera cases and ϳ50% of all essential thrombocythemia and primitive myelofibrosis cases (3, 4). Several additional mutations leading to constitutively active JAK2 have been identified recently in patients, including a JAK2 K539L mutant. A T875N substitution in JAK2 was also identified in megakaryoblastic myeloid leukemia cells. In a patient with trisomy 21 and B cell precursor acute lymphoblastic l...

Despite efforts to discover the cellular pathways regulating breast cancer metastasis, little is known as to how prolactin (PRL) cooperates with extracellular environment and cytoskeletal proteins to regulate breast cancer cell motility and invasion. We implicated serine-threonine kinase p21-activated kinase 1 (PAK1) as a novel target for PRL-activated Janus-kinase 2 (JAK2). JAK2-dependent PAK1 tyrosyl phosphorylation plays a critical role in regulation of both PAK1 kinase activity and scaffolding properties of PAK1. Tyrosyl phosphorylated PAK1 facilitates PRL-dependent motility via at least two mechanisms: formation of paxillin/GIT1/βPIX/pTyr-PAK1 complexes resulting in increased adhesion turnover and phosphorylation of actin-binding protein filamin A. Increased adhesion turnover is the basis for cell migration and phosphorylated filamin A stimulates the kinase activity of PAK1 and increases actin-regulating activity to facilitate cell motility. Tyrosyl phosphorylated PAK1 also stimulates invasion of breast cancer cells in response to PRL and three-dimensional (3D) collagen IV via transcription and secretion of MMP-1 and MMP-3 in a MAPK-dependent manner. These data illustrate the complex interaction between PRL and the cell microenvironment in breast cancer cells and suggest a pivotal role for PRL/PAK1 signaling in breast cancer metastasis.

The p21-activated serine-threonine kinase (PAK1) regulates cell motility and adhesion. We have previously shown that the prolactin (PRL)-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo and in vitro and identified tyrosines 153, 201, and 285 in PAK1 as sites of JAK2 tyrosyl phosphorylation. Here, we further investigate the role of the tyrosyl phosphorylated PAK1 (pTyr-PAK1) in regulation of cell adhesion. We use human breast cancer T47D cell lines that stably overexpress PAK1 wild type or PAK1 Y3F mutant in which these 3 JAK2 phosphorylation sites were mutated to phenylalanine. We demonstrate that PRL/JAK2-dependent phosphorylation of these tyrosines promotes a motile phenotype in the cells upon adhesion, participates in regulation of cell adhesion on collagen IV, and is required for maximal PAK1 kinase activity. Down-regulation of PAK1 abolishes the effect of PAK1 on cell adhesion. We show that the tyrosyl phosphorylation of PAK1 promotes PAK1 binding to β-PAK1-interacting guanine-nucleotide exchange factor (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), phosphorylation of paxillin on Ser273, and formation and distribution of adhesion complexes. Using phosphospecific antibodies (Abs) directed to single phosphorylated tyrosines on PAK1, we identified Tyr285 as a site of PRL-dependent phosphorylation of PAK1 by JAK2. Furthermore, using PAK1 Y285F mutant, we provide evidence for a role of pTyr285 in cell adhesion, enhanced βPIX/GIT1 binding, and adhesion turnover. Our immunohistochemistry analysis demonstrates that pTyr285- PAK1 may modulate PAK1 signaling during tumor progression.

BackgroundThe serine/threonine kinase PAK1 is an important regulator of cell motility. Both PAK1 and the hormone/cytokine prolactin (PRL) have been implicated in breast cancer cell motility, however, the exact mechanisms guiding PRL/PAK1 signaling in breast cancer cells have not been fully elucidated. Our lab has previously demonstrated that PRL-activated tyrosine kinase JAK2 phosphorylates PAK1 on tyrosines 153, 201, and 285, and that tyrosyl phosphorylated PAK1 (pTyr-PAK1) augments migration and invasion of breast cancer cells.ResultsHere we further investigate the mechanisms by which pTyr-PAK1 enhances breast cancer cell motility in response to PRL. We demonstrate a distinct reduction in PRL-induced FAK auto-phosphorylation in T47D and TMX2-28 breast cancer cells overexpressing wild-type PAK1 (PAK1 WT) when compared to cells overexpressing either GFP or phospho-tyrosine-deficient mutant PAK1 (PAK1 Y3F). Furthermore, pTyr-PAK1 phosphorylates MEK1 on Ser298 resulting in subsequent ERK1/2 activation. PRL-induced FAK auto-phosphorylation is rescued in PAK1 WT cells by inhibiting tyrosine phosphatases and tyrosine phosphatase inhibition abrogates cell motility and invasion in response to PRL. siRNA-mediated knockdown of the tyrosine phosphatase PTP-PEST rescues FAK auto-phosphorylation in PAK1 WT cells and reduces both cell motility and invasion. Finally, we provide evidence that PRL-induced pTyr-PAK1 stimulates tumor cell metastasis in vivo.ConclusionThese data provide insight into the mechanisms guiding PRL-mediated breast cancer cell motility and invasion and highlight a significant role for pTyr-PAK1 in breast cancer metastasis.