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Biomedical subjects

Ratna K Vadlamudi

Publications and source records attributed to Ratna K Vadlamudi.

At least 19 recordsLinked to original sources

Antibody-Mediated Targeting of Secretory Protein SCUBE3 Suppresses Cancer Progression by Inhibiting Oncogenic Signaling and Inducing Antitumor Immunity.

UNLABELLED: Approaches targeting factors that simultaneously promote tumor growth and progression, induce therapy resistance, and inhibit antitumor immunity offer clear benefits over therapies targeting only one of these tumor-promoting processes. Through comprehensive loss-of-function genomic screening, we identified SCUBE3 as a pivotal factor that supports survival and therapy resistance and also orchestrates an immunosuppressive tumor microenvironment. Secretory SCUBE3 supported oncogenic activity through interactions with key oncogenic cell surface receptor proteins, including EGFR, mutant CALR, and TGFβRI/II. These interactions activated the transcription factors FOXR2 and c-Myc, promoting cancer cell proliferation and therapy resistance by enhancing DNA damage repair. Additionally, the SCUBE3-FOXR2 axis created an immunosuppressive tumor microenvironment by facilitating recruitment of the DNMT1 epigenetic repressor complex to the transcription regulator IRF1, thereby inhibiting the expression of MHC-I and MHC-II genes. A first-in-class neutralizing antibody targeting SCUBE3, which was developed using a sophisticated antibody discovery platform and engineered with specific mutations in the heavy chain for enhanced specificity and efficacy, demonstrated profound therapeutic potential across various cancer types in preclinical models, including patient-derived breast and ovarian cancer xenografts. This discovery marks an advancement toward developing a targeted therapy for cancers characterized by hyperactive SCUBE3-associated signaling pathways. SIGNIFICANCE: Targeting SCUBE3 with a neutralizing antibody inhibits tumor growth and metastasis by blocking oncogenic signaling through FOXR2 and c-Myc and by circumventing immunosuppression, providing a promising pan-cancer treatment approach.

Humans↗

Proline-, glutamic acid-, and leucine-rich protein-1/modulator of nongenomic activity of estrogen receptor enhances androgen receptor functions through LIM-only coactivator, four-and-a-half LIM-only protein 2.

Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a coregulator of multiple nuclear receptors. Molecular mechanisms of PELP1 function are not completely understood, but its expression is up-regulated in hormonal-dependent cancers. Using a yeast two-hybrid screen, we found that four-and-a-half LIM-only protein 2 (FHL2) interacted with PELP1. FHL2 is a transcriptional regulator that associates with nuclear cofactors, including androgen receptors (ARs), and contains an intrinsic activation domain. PELP1 and FHL2 interact in vitro and in vivo and colocalize in the nuclear compartment. PELP1 interacts with FHL2 via LIM domains 3 and 4 and synergistically enhances the transcriptional activity of FHL2. Src kinase is required for PELP1-mediated enhancement of FHL2 functions because knockdown of Src kinase expression or function abolished PELP1-mediated FHL2 activation functions. PELP1 interacted with AR and enhanced FHL2-mediated AR transactivation functions. PELP1 knockdown by small interfering RNA or PELP1 mutant, which lacks an activation domain, reduced FHL2-mediated AR transactivation. Biochemical analyses revealed a complex consisting of PELP1, FHL2, and AR in prostate cancer cells. PELP1/MNAR expression was elevated in high-grade prostate tumors. Our results suggest that PELP1 functions as a molecular adaptor, coupling FHL2 with nuclear receptors, and PELP1-FHL2 interactions may have a role in prostate cancer progression.

Animals↗

Coregulation of estrogen receptor by ERBB4/HER4 establishes a growth-promoting autocrine signal in breast tumor cells.

Although crosstalk between cell-surface and nuclear receptor signaling pathways has been implicated in the development and progression of endocrine-regulated cancers, evidence of direct coupling of these signaling pathways has remained elusive. Here we show that estrogen promotes an association between extranuclear estrogen receptor alpha (ER) and the epidermal growth factor receptor (EGFR) family member ERBB4. Ectopically expressed as well as endogenous ERBB4 interacts with and potentiates ER transactivation, indicating that the ERBB4/ER interaction is functional. Estrogen induces nuclear translocation of the proteolytic processed ERBB4 intracellular domain (4ICD) and nuclear translocation of 4ICD requires functional ligand-bound ER. The nuclear ER/4ICD complex is selectively recruited to estrogen-inducible gene promoters such as progesterone receptor (PgR) and stromal cell-derived factor 1 (SDF-1) but not to trefoil factor 1 precursor (pS2). Consistent with 4ICD-selective promoter binding, suppression of ERBB4 expression by interfering RNA shows that 4ICD coactivates ER transcription at the PgR and SDF-1 but not the pS2 promoter. Significantly, ERBB4 itself is an estrogen-inducible gene and the ERBB4 promoter harbors a consensus estrogen response element (ERE) half-site with overlapping activator protein-1 elements that bind ER and 4ICD in response to estrogen. Using a cell proliferation assay and a small interfering RNA approach, we show that ERBB4 expression is required for the growth-promoting action of estrogen in the T47D breast cancer cell line. Our results indicate that ERBB4 is a unique coregulator of ER, directly coupling extranuclear and nuclear estrogen actions in breast cancer. We propose that the contribution of an autocrine ERBB4/ER signaling pathway to tumor growth and therapeutic response should be considered when managing patients with ER-positive breast cancer.

Breast Neoplasms↗

Comprehensive analysis of recent biochemical and biologic findings regarding a newly discovered protein-PELP1/MNAR.

Estradiol (E2) and estrogen receptor (ER) signaling have been implicated in the development and progression of several cancers. Emerging evidence suggests that the status of ER coregulators in tumor cells plays an important role in hormonal responsiveness and tumor progression. Proline, glutamic acid, and leucine-rich protein-1 (PELP1/MNAR)-a novel ER coactivator that plays an essential role in the ER's actions and its expression-is deregulated in several hormonal responsive cancers. The precise function of PELP1/MNAR in cancer progression remains unclear, but PELP1 appears to function as a scaffolding protein, coupling ER with several proteins that are implicated in oncogenesis. Emerging evidence suggests that PELP1/MNAR increases E2-mediated cell proliferation and participates in E2-mediated tumorigenesis and metastasis.

Breast Neoplasms↗

9-cis-retinoic acid up-regulates expression of transcriptional coregulator PELP1, a novel coactivator of the retinoid X receptor alpha pathway.

Retinoid X receptor alpha (RXRalpha), functioning as either a homodimer or a heterodimer with peroxisome proliferator receptors, is known to be involved in manifesting antiproliferative effects in cells. Consequently, studies of RXRalpha functions and its coregulators have been in the focus for therapeutic approaches against cancer. Here we have discovered that 9-cis-retinoic acid (9-cis-RA), a RXRalpha-specific ligand, up-regulated the expression of transcriptional coregulatory protein PELP1 (proline-, glutamic acid-, and leucine-rich protein 1). PELP1 functioned as a coactivator of RXRalpha, increasing its transactivation function in response to 9-cis-RA as evident by the retinoid X receptor response element-luciferase assays. PELP1 was found to be a binding partner of RXRalpha, and the binding interactions were confirmed both in vitro and in vivo. An electrophoretic mobility shift assay showed greater formation and stability of RXRalpha homodimers on consensus oligonucleotides in PELP1-overexpressing clones in comparison to the pcDNA clones. The presence of PELP1 in these oligonucleotide-bound RXRalpha homodimers was proved by the supershift of the complex when incubated with PELP1-specific antibody. PELP1-overexpressing stable MCF-7 cells exhibited a significantly higher extent of 9-cis-RA-induced apoptosis than the control pcDNA clones. Silencing of PELP1 expression in parental MCF-7 cells and PELP1-overexpressing clones using PELP1-specific RNA-mediated interference compromised the susceptibility to 9-cis-RA-induced apoptosis. PELP1 could also function as a coactivator of the RXRalpha-peroxisome proliferator-activated receptor (PPARgamma) heterodimer as evident by the peroxisome proliferator-activated receptor response element-luciferase assay in response to both 9-cis-RA and PPARgamma-specific ligands. This was reinforced by the higher propensity of PELP1-overexpressing clones to undergo differentiation in response to PPARgamma-specific ligands. This study has revealed a novel facet of PELP1 functions and identified it to be an important potentiator of the antiproliferative effects of 9-cis-RA and PPARgamma-specific ligands.

Alitretinoin↗

Novel mechanisms of resistance to endocrine therapy: genomic and nongenomic considerations.

Selective estrogen receptor (ER) modulators have been the most commonly used neoadjuvant therapy for hormone-dependent breast cancer. However, resistance to endocrine therapy, either inherent or acquired during treatment, presents a major challenge in disease management. The causes of resistance to hormone therapy are not well understood and are the subject of active investigation. It is increasingly clear that decreasing sensitivity of ER-positive breast cancer cells to antiestrogens is caused by several factors. Cross talk between ER and growth factor signaling has emerged as a critical factor in endocrine resistance. Here, we present evidence that receptor tyrosine kinase signaling also plays a role in resistance by controlling the subcellular localization of ER signaling components. Localization of ER in either the nuclear or cytoplasmic compartments has functional implications. Recent work suggests that dynein light chain 1, a recently identified substrate of p21-activated kinase 1, modulates ER transactivation functions through a novel ER coactivator function. Likewise, receptor tyrosine kinase signaling can also alter the expression of ER coregulators such as metastasis-associated antigen 1, leading to hormonal independence. Furthermore, proline-, glutamic acid-, leucine-rich protein 1, an ER coactivator involved in both genomic and nongenomic signaling pathways, is activated by epidermal growth factor receptor and plays a prominent role in resistance to tamoxifen. These recent advances suggest new targeted therapeutic approaches that may lead to either reversion or prevention of endocrine resistance in breast tumors.

Animals↗

Altered localization of a coactivator sensitizes breast cancer cells to tumor necrosis factor-induced apoptosis.

Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel coregulator of the estrogen receptor that plays a role in both genomic and nongenomic actions of the estrogen receptor. Emerging studies suggest that in addition to the nuclear localization of PELP1, it is predominantly localized in the cytoplasm in human breast tumors, leading to excessive nongenomic signaling and possibly to tamoxifen resistance. The mechanisms underlying resistance to hormones in preclinical model systems remain under intense investigation. In an effort to develop a model system to treat tumor cells with cytoplasmic PELP1 expression and tamoxifen resistance, here we used the cytokine tumor necrosis factor (TNF)-alpha. We found that clones of MCF-7 human breast cancer cells overexpressing PELP1 in the cytoplasm were distinctly sensitive to TNF-alpha-induced apoptosis than were wild-type nuclear PELP1- and pcDNA vector-expressing clones as revealed by cell growth assay, cell cycle analysis, Annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. We also found that the clones with cytoplasmic PELP1 overexpression had significantly less antiapoptotic protein Bcl-2 and nuclear factor-kappaB DNA binding, but increased cyclin E expression, further supporting evidence that these cells are sensitive to apoptosis. The mechanism behind TNF-induced apoptosis in these cells involves caspases, as revealed by poly(ADP-ribose) polymerase cleavage and the broad-spectrum caspase inhibitor Z-VAD-inhibited apoptosis. In conclusion, our results suggest that altered localization of PELP1 promotes heightened sensitivity to TNF-alpha in MCF-7 cells, paving the way for developing new treatment strategies for tumors with cytoplasmic PELP1 expression.

Apoptosis↗

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacts with PELP1 and activates MAPK.

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) (also known as the modulator of nongenomic activity of estrogen receptor) plays a role in genomic functions of the estrogen receptor via histone interactions and in nongenomic functions via its influence on the MAPK-Src pathway. However, recent studies have shown that differential compartmentalization of PELP1 could play a crucial role in modulating the status of nongenomic signaling by using molecular mechanisms that remain poorly understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is an early endosomal protein that plays a role in regulating the trafficking of growth factor-receptor complexes through early endosomes. By using a yeast two-hybrid screen, we identified HRS as a novel PELP1-binding protein providing evidence of a physiologic interaction between HRS and PELP1. The noted HRS-PELP1 interaction was accompanied by inhibition of the basal coactivator function of PELP1 upon estrogen receptor transactivation. HRS was found to sequester PELP1 in the cytoplasm, leading to the activation of MAPK in a manner that is dependent on the epidermal growth factor receptor but independent of the estrogen receptor, Shc, and Src. In addition, stimulation of MAPK and the subsequent activation of its downstream effector pathway, Elk-1, by HRS or PELP1 were found to depend on the presence of endogenous PELP1 or HRS. Furthermore, HRS was overexpressed and correlated well with the cytoplasmic PELP1, increased MAPK, and EGFR status in breast tumors. These findings highlight a novel role of HRS in up-regulating MAPK, presumably involving interaction with PELP1.

Animals↗

Functional implications of altered subcellular localization of PELP1 in breast cancer cells.

It is increasingly accepted that steroidal receptor co-regulators may also function in the cytoplasmic compartment. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel coregulator that plays a role in both the genomic and extranuclear actions of estrogen receptors (ER) in hormonally responsive tissues. In this study using breast tumor arrays, we found that PELP1 was localized only in the cytoplasm in 58% of the PELP1-positive breast tumors. To help explain the significance of the cytoplasmic localization of PELP1 in human breast tumors, we created a mutant protein that was expressed only in the cytoplasm (PELP1-cyto) and then generated a model system wherein MCF-7 breast cancer cells were engineered to specifically express this mutant. We found that PELP1-cyto cells were hypersensitive to estrogen but resistant to tamoxifen. PELP1-cyto cells, but not parental MCF-7 cells, formed xenograft tumors in nude mice. In addition, PELP1-cyto cells exhibited increased association of PELP1 with Src, enhanced mitogen-activated protein kinase (MAPK) activation, and constitutive activation of AKT. The altered localization of PELP1 was sufficient to trigger the interaction of PELP1 with the p85 subunit of phosphatidylinositol-3-kinase (PI3K), leading to PI3K activation. In addition, PELP1 interacted with epidermal growth factor receptors and participated in growth factor-mediated ER transactivation functions. Our results suggest that the altered localization of PELP1 modulates sensitivity to antiestrogens, potentiates tumorigenicity, presumably via the stimulation of extranuclear estrogen responses, such as the activation of MAPK and AKT, and also enhance cross-regulation of ER transactivation activity by growth factors.

Animals↗

Cloning, expression, and localization of MNAR/PELP1 in rodent brain: colocalization in estrogen receptor-alpha- but not in gonadotropin-releasing hormone-positive neurons.

MNAR/PELP1 is a recently identified scaffold protein in the human that modulates the nongenomic activity of estrogen receptors by facilitating linkage/cross talk with the Src/Erk activation cascade. We report herein the cloning of rat MNAR/PELP1 and provide new information concerning its distribution in the female rat brain and its degree of colocalization with estrogen receptor-alpha (ER-alpha) and GnRH. PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence and retained all the key binding motifs (PXXP, LXXLL, and glutamic acid clusters) in its primary structure that are known to be critical for its interaction with Src and steroid receptors. RT-PCR revealed that the MNAR/PELP1 transcript is expressed in many regions of the brain, and immunohistochemistry studies showed intense MNAR/PELP1 immunoreactivity (MNAR/PELP1-ir) in areas such as the hypothalamus, cerebral cortex, hippocampus, amygdala, and cerebellum. MNAR/PELP1-ir principally localized in the nucleus, but some cytoplasmic and plasma membrane-associated staining was also observed. MNAR/PELP1-ir was also primarily neuronal, although some localization in glia cells was observed in select brain regions. Colocalization studies revealed that a majority of ER-alpha-positive cells in the brain colocalized MNAR/PELP1-ir. In contrast, MNAR/PELP1-ir rarely colocalized in GnRH neurons. In conclusion, the current study provides evidence that MNAR/PELP1 is expressed in key neural tissues of the rat brain that are known targets of steroid action, that its expression is primarily neuronal, and that MNAR/PELP1-ir is strongly colocalized in ER-alpha, but not GnRH neurons in the rodent brain.

Animals↗

Proline-, glutamic acid-, and leucine-rich protein-1 is essential in growth factor regulation of signal transducers and activators of transcription 3 activation.

Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) is a novel estrogen receptor coactivator that plays an important role in the genomic and nongenomic actions of estrogen receptor by interacting with histones and src-mitogen-activated protein kinase pathway, respectively. A great deal of information has emerged in recent years about the possible role of PELP1 in estrogen receptor signaling. However, the participation and significance of PELP1 in other cellular signaling pathways remains unknown. Using a yeast two-hybrid screen, we identified PELP1 as a novel interacting protein of signal transducers and activators of transcription 3 (STAT3) and found evidence of physiologic interaction between PELP1 and STAT3. We also found that these interactions played a mechanistic role in the positive regulation of STAT3 transcription from synthetic promoters and endogenous target genes such as cyclin D1, c-myc, and c-fos. Overexpression of PELP1 enhanced phosphorylation of STAT3 at Ser727 in a src-mitogen-activated protein kinase-sensitive manner and, conversely, down-regulation of PELP1 compromised growth factor-mediated induction of STAT3 target genes. We also discovered that PELP1 interacts with STAT3 in the nuclear compartment and down-regulation of PELP1 interfered with the recruitment of STAT3 to its target gene promoters. In summary, our results highlight a novel role for PELP1 in growth factor signaling and indicate that PELP1-mediated genomic and nongenomic functions play a role in the growth factor-mediated STAT3 transactivation functions. Such regulatory interactions of PELP1 may have important functional implications in the cross-talk of estrogen receptor and growth factor signaling.

Animals↗

An essential role of Pak1 phosphorylation of SHARP in Notch signaling.

The p21-activated kinases (Paks), an evolutionarily conserved family of serine/threonine kinases, play an important role in cytoskeletal reorganization in mammalian cells. The Notch signaling pathway plays an important role in the determination of cell fate/differentiation in a number of organs. Notch signaling is a complex process, and the mechanism by which Notch regulates multiple cellular processes is intriguing. The expression of both Notch and Pak1 has been shown to be deregulated in several human cancers. Using yeast two-hybrid screening, we identified SHARP, one of the Notch signaling components, as a Pak1-interacting protein. We found that SHARP is a physiologic interacting substrate of Pak1, and that this interaction enhances SHARP-mediated repression of Notch target genes. Pak1 phosphorylation sites in SHARP were mapped to Ser3486 and Thr3568 within the SHARP repression domain. Mutation of Pak1 phosphorylation sites in SHARP, inhibition of Pak1 functions by a Pak1-autoinhibitory fragment (amino acids 83-149), or expression of Pak1-specific siRNA interfered with SHARP-mediated repression of Notch target reporter gene activation. These results demonstrate that Pak1-SHARP interaction plays an essential role in enhancing the corepressor functions of SHARP, thereby modulating Notch signaling in human cancer cells.

3T3 Cells↗

Pak1 phosphorylation of snail, a master regulator of epithelial-to-mesenchyme transition, modulates snail's subcellular localization and functions.

The process of epithelial-mesenchymal transition plays a pivotal role in the conversion of early stage tumors into invasive malignancies, and has been shown to be regulated by the zinc finger phosphoprotein, Snail; however, no upstream signaling kinases have been shown to modulate Snail functions. Since the invasiveness of breast cancer cells is also influenced by p21-activated kinase 1 (Pak1) signaling, we investigated Pak1's potential mechanistic role in the regulation of Snail functions. We found for the first time that Pak1 promotes transcription repression activity of Snail from E-cadherin, occludin, and aromatase promoters. Pak1 regulates the repressor activity of Snail by phosphorylating on Ser(246). Pak1 phosphorylation of Snail supports Snail's accumulation in the nucleus as well as its repressor functions. A Ser(246)Ala substitution in Snail or Pak1 knockdown by short interference RNA blocked Pak1-mediated Snail phosphorylation, leading to increased cytoplasmic accumulation of Snail and attenuation of Snail repressor activity in breast cancer cells. The regulation of phosphorylation and function of Snail by Pak1 represents a novel mechanism by which a signaling kinase might contribute to the process of epithelial-mesenchymal transition.

Amino Acid Sequence↗

The clinical relevance of steroid hormone receptor corepressors.

Steroid hormone receptors are ligand-dependent transcription factors that control a variety of essential physiologic and developmental processes in humans. The functional activity of a steroid receptor is regulated not only by hormones but also by an array of regulatory proteins such as coactivators, corepressors, and chromatin modifiers. Contrary to an earlier notion that corepressors and coactivators exist in separate complexes, these molecules, which have apparently opposite functions, are increasingly being found in the same complex, which allows for efficient transcriptional control mechanisms. These control mechanisms are in turn regulated by an array of post-translational modifications under the influence of upstream and local signaling networks. Because the outcome of steroidal hormone receptor transcriptional complexes is measured in terms of the expression of target genes, any dysregulation of coregulator complexes perturbs normal homeostasis and could contribute to the development and maintenance of malignant phenotypes. Increasing evidence implicating steroid hormone receptors and their coregulators in various pathophysiologic conditions has elicited interest in their structure and biology. Further advances in this field of study should open up a unique window for novel targeted therapies for diseases such as cancer. Here we briefly review the clinical relevance of corepressors, with a particular focus on their role in the development of cancerous phenotypes.

DNA-Binding Proteins↗

Novel estrogen receptor coactivator PELP1/MNAR gene and ERbeta expression in salivary duct adenocarcinoma: potential therapeutic targets.

Salivary duct carcinoma (SDC) is a high-grade neoplasm with marked morphological resemblance to mammary duct carcinoma. The novel estrogen receptor (ER)-interacting protein and the proline-, glutamic acid-, and leucine-rich protein 1 ( PELP1 ), also called the modulator of nongenomic activity of ER ( MNAR ), have been shown to activate steroid hormone receptors in mammary carcinomas by nongenomic and genomic mechanisms. The expression and the relationship of this gene to the ER status in SDCs are unknown. We investigated the differential expression of the PELP1 / MNAR and the ERs alpha and beta proteins in SDCs, using Western blotting and immunohistochemistry. Western blot analysis of 7 paired normal and tumor specimens showed increased expression of PELP1 / MNAR and ER beta in 3 and 4 of the SDCs, respectively. No detectable expression of ER alpha in any normal or SDC specimens was noted. Immunohistochemical staining performed on 70 SDCs revealed strong expression of PELP1 / MNAR in 51 (73%) and ER beta in 52 (74%) tumors. PELP1 / MNAR and ER beta were coexpressed in 35 (50%), individually in 17 (24.2%), and negative in 18 (25.7%) tumors. PELP1 / MNAR staining was predominantly cytoplasmic whereas ER beta staining was nuclear and occasionally cytoplasmic in tumor cells. Our results indicate that PELP1 / MNAR and ER beta are coexpressed in most SDCs and may play a role in the pathobiology of these tumors.

Adenocarcinoma↗

p21-activated kinase 1 regulates microtubule dynamics by phosphorylating tubulin cofactor B.

p21-activated kinase 1 (Pak1) induces cytoskeleton reorganization in part by regulating microtubule dynamics through an elusive mechanism. Using a yeast two-hybrid screen, we identified tubulin cofactor B (TCoB) (a cofactor in the assembly of the alpha/beta-tubulin heterodimers) as an interacting substrate of Pak1. Pak1 directly phosphorylated TCoB in vitro and in vivo on serines 65 and 128 and colocalized with TCoB on newly polymerized microtubules and on centrosomes. TCoB interacted with the GTPase-binding domain of Pak1 and activated Pak1 in vitro and in vivo. In contrast to wild-type TCoB, an S65A, S128A double mutant and knock-down of the endogenous TCoB or Pak1 reduced microtubule polymerization, suggesting that Pak1 phosphorylation is necessary for normal TCoB function. Overexpression of TCoB dramatically increased the number of gamma-tubulin-containing microtubule-organizing centers, a phenotype reminiscent of cells overexpressing Pak1. TCoB was overexpressed and phosphorylated in breast tumors. These findings reveal a novel role for TCoB and Pak1 in regulating microtubule dynamics.

Breast Neoplasms↗

Dynein light chain 1 phosphorylation controls macropinocytosis.

Recent studies have identified dynein light chain-1 (DLC1), a component of the dynein motor, as a p21-activated kinase 1 (Pak1)-interacting substrate with binding sites mapped to amino acids 61-89 of DLC1 and phosphorylation site at serine 88. Here we investigated the role of DLC1 phosphorylation by Pak1 upon the process of macropinocytosis. We found that Pak1 associates with dynein motor and that Pak1-DLC1 interaction starts at the initiation of pinosome formation and persists in early and late endosomes. Pak1 phosphorylation of DLC1 on Ser-88 controls vesicle formation and trafficking functions, as Ser-88 substitution for alanine prevents macropinocytosis. A peptide spanning the C-terminal 19-amino acid region of DLC1 efficiently blocked Ser-88 phosphorylation and macropinocytosis. These results suggest that the regulation of DLC1 by Pak1 is a novel mechanism by which a signaling kinase might influence macropinocytosis.

Animals↗

Regulation of phosphoglucomutase 1 phosphorylation and activity by a signaling kinase.

We have identified a novel mechanism of cross-talk between cell signaling and metabolic pathways, whereby the signaling kinase p21-activated kinase 1 (Pak1) binds to, phosphorylates and enhances the enzymatic activity of phosphoglucomutase 1 (PGM), an important regulatory enzyme in cellular glucose utilization and energy homeostasis. Pak1 and PGM were colocalized in model cell systems and showed functional interactions in a physiological setting. Strong direct interaction of PGM with Pak1 but not Pak2, Pak3, or Pak4 was observed. PGM binding was within 75-149 amino acids (aa) of Pak1, while Pak1 binding to PGM was in the N-terminal 96 aa. Pak1-mediated phosphorylation of PGM selectively on threonine 466 significantly increased PGM enzymatic activity and could be blocked by transfection with a dominant-negative Pak1 expression vector and by Pak1-specific small inhibitory RNA. Stable transfection of PGM into PGM-deficient K562 leukemia cells further demonstrated the role of Pak1 in regulating PGM activity. The results presented here provide new evidence that the cell signaling kinase Pak1 is a novel regulator of glucose metabolism through its phosphorylation and regulation of PGM activity. These findings suggest a new mechanism whereby growth factor signaling may coordinately integrate metabolic regulation with established signaling functions of cell cycle regulation and cell growth.

Amino Acid Sequence↗