Irwin H. Gelman

SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.

A Novel src- and ras-suppressed Protein Kinase C Substrate Associated with Cytoskeletal Architecture

We previously identified a novel src- and ras-suppressed gene, 322, encoding a mitogenic regulatory function (Lin, X., Nelson, P. J., Frankfort, B., Tombler, E., Johnson, R., and Gelman, I. H. (1995) Mol. Cell. Biol. 15, 2754-2762). Here, we characterize the 322 gene product as an in vivo and in vitro substrate of protein kinase C (PKC). Hence, we named this product SSeCKS (pronounced essex) for Src Suppressed C Kinase Substrate. Rabbit polyclonal sera raised against glutathione S-transferase (GST)-SSeCKS recognized a myristylated 280/290-kDa doublet in Rat-6 fibroblasts. SSeCKS levels in src- and ras-transformed Rat-6 cells were 15- and 8-fold less, respectively, than those in untransformed cells. Short-term addition of phorbol ester resulted in a 5-fold increase in SSeCKS phosphorylation which was inhibited by bis-indolylmaleimide. In vitro phosphorylation of GST-SSeCKS by purified rabbit brain PKC-α was enhanced by phosphatidylserine and blocked by excess PKC pseudosubstrate inhibitor peptide. GST-SSeCKS bound purified PKC-α or PKC from Rat-6 lysates in a phosphatidylserine-dependent manner. Four SSeCKS domains containing Lys/Arg-rich motifs similar to the PKC phosphorylation site in MARCKS were phosphorylated in vitro by PKC. Immunofluorescence analysis showed SSeCKS present throughout the cytoplasm with enrichment in podosomes and at the cell edge. Short-term addition of phorbol esters caused the movement of SSeCKS from plasma membrane sites to the perinucleus coincident with a loss of actin stress fibers. These data suggest a role for SSeCKS in the control of cellular cytoskeletal architecture.

SSeCKS, a Major Protein Kinase C Substrate with Tumor Suppressor Activity, Regulates G1→S Progression by Controlling the Expression and Cellular Compartmentalization of Cyclin D

ABSTRACT SSeCKS, first isolated as a G1→S inhibitor that is downregulated in src- and ras-transformed cells, is a major cytoskeleton-associated PKC substrate with tumor suppressor and kinase-scaffolding activities. Previous attempts at constitutive expression resulted in cell variants with truncated ectopic SSeCKS products. Here, we show that tetracycline-regulated SSeCKS expression in NIH 3T3 cells induces G1 arrest marked by extracellular signal-regulated kinase 2-dependent decreases in cyclin D1 expression and pRb phosphorylation. Unexpectedly, the forced reexpression of cyclin D1 failed to rescue SSeCKS-induced G1 arrest. Confocal microscopy analysis revealed cytoplasmic colocalization of cyclin D1 with SSeCKS. Because the SSeCKS gene encodes two potential cyclin-binding motifs (CY) flanking major in vivo protein kinase C (PKC) phosphorylation sites (Ser507/515), we addressed whether SSeCKS encodes a phosphorylation-dependent cyclin scaffolding function. Bacterially expressed SSeCKS-CY bound cyclins D1 and E, whereas K→S mutations within either CY motif ablated binding. Activation of PKC in vivo caused a rapid translocation of cyclin D1 to the nucleus. Cell permeable, penetratin-linked peptides encoding wild-type SSeCKS-CY, but not K→S or phospho-Ser507/515 variants, released cyclin D1 from its cytoplasmic sequestration and induced higher saturation density in cyclin D1-overexpressor cells or rat embryo fibroblasts. Our data suggest that SSeCKS controls G1→S progression by regulating the expression and localization of cyclin D1. These data suggest that downregulation of SSeCKS in tumor cells removes gating checkpoints for saturation density, an effect that may promote contact independence.

ISOLATION AND CHARACTERIZATION OF A NOVEL MITOGENIC REGULATORY GENE, 322, WHICH IS TRANSCRIPTIONALLY SUPPRESSED IN CELLS TRANSFORMED BY SRC AND RAS

In an attempt to isolate novel regulatory and/or tumor suppressor genes, we identified cDNAs whose abundance is low in NIH 3T3 cells and further decreased following the expression of the activated oncogene, v-src. The transcription of one such gene, 322, is suppressed at least 15-fold in src-, ras-, and fos-transformed cells and 3-fold in myc-transformed cells but is unaffected in raf-, mos-, or neu-transformed cells. Activation of a ts-v-src allele in confluent 3Y1 fibroblasts resulted in an initial increase in 322 mRNA levels after 1 to 2 h followed by a rapid decrease to suppressed levels after 4 to 8 h. Morphological transformation was not detected until 12 h later, indicating that the accumulation of 322 transcripts is regulated by v-src and not as a consequence of transformation. Addition of fetal calf serum to starved subconfluent NIH 3T3 or 3Y1 fibroblasts resulted in a similar biphasic regulation of 322, indicating that 322 transcription is responsive to mitogenic factors. Sequence analysis of a putative full-length 322 cDNA clone (5.4 kb) identified a large open reading frame (ORF) encoding a 148.1-kDa product. In vitro transcription and translation of the 322 cDNA from a T7 promoter resulted in a 207-kDa product whose electrophoretic mobility on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel was unaffected by digestion with endoglycosidase F. The discrepancy in predicted versus measured molecular weights may result from the high percentage of acidic residues (roughly 20% Glu or Asp) in the 322 ORF product. Comparison of the 322 cDNA ORF with sequences in data banks indicates that this gene is novel. The 322 ORF product contains a potential Cys-1-His-3 Zn finger, at least five nuclear localization signals of the adenovirus E1a motif K(R/K)X(R/K), and alternating acidic and basic domains. Overexpression of the 322 cells resulted in the selection of rapidly growing cells which had lost the transduced 322 cDNA. Thus, 322 represent a novel src- and ras-regulated gene which encodes a potential regulator of mitogenesis and/or tumor suppressor.

CONTROL OF CYTOSKELETAL ARCHITECTURE BY THE SRC-SUPPRESSED C KINASE SUBSTRATE, SSECKS

Activation of protein kinase C (PKC) in many cell types results in cytoskeletal reorganization associated with cell proliferation. We previously described a new cell cycle-regulated myristylated PKC substrate, SSeCKS (pronounced essex), that interacts with the actin cytoskeleton [Lin et al., 1995, 1996]. SSeCKS shares significant homology with Gravin, which encodes kinase scaffolding functions for PKC and PKA [Nauert et al., 1997]. This article describes the cellular effects of ectopically expressing SSeCKS in untransformed NIH3T3 fibroblasts. Because the constitutive overexpression of SSeCKS is toxic [Lin et al., 1995], we developed cell lines with tetracycline (tet)-regulated SSeCKS expression. The induction of SSeCKS (removal of tet) caused significant cell flattening and the elaboration of an SSeCKS-associated cortical cytoskeletal matrix resistant to Triton X-100 extraction. Flattened cells were growth-arrested and marked by the formation of cellular projections and the temporary loss of actin stress fibers and vinculin-associated adhesion plaques. SSeCKS overexpression did not affect steady-state levels of actin, vinculin, or focal adhesion kinase (FAK) but did increase integrin-independent FAK tyrosine phosphorylation. Stress fiber loss was coincident with induced SSeCKS expression, strongly suggesting a direct effect. Cytochalasin, and to a lesser extent nocodazole, inhibited SSeCKS-induced cell flattening, however, only cytochalasin affected the shape of pre-flattened cells, suggesting a greater dependence on microfilaments, rather than microtubules. By contrast, only nocodazole caused retraction of the filopodia-like processes. These data indicate a role for SSeCKS in modulating both cytoskeletal and signaling pathways. Thus, we propose to expand SSeCKS scaffolding functions to include the ability to control actin-based cytoskeletal architecture, as well as mitogenic signal pathways.

A Role for SSeCKS, a Major Protein Kinase C Substrate with Tumour Suppressor Activity, in Cytoskeletal Architecture, Formation of Migratory Processes, and Cell Migration During Embryogenesis

SSeCKS is a major protein kinase C substrate which has tumour suppressor activity in models of src- and ras-induced oncogenic transformation. The mitogenic regulatory activity of SSeCKS is likely manifested by its ability to bind key signalling proteins such as protein kinases C and A and calmodulin, and to control actin-based cytoskeletal architecture. Rat SSeCKS shares extensive homology with human Gravin, an autoantigen in myasthenia gravis that encodes kinase scaffolding functions and whose expression pattern in fibroblasts and nerves suggests a role in cell motility. Here, we analyse the expression of SSeCKS and Gravin in rodent and human fibroblast and epithelial cell lines using antibodies specific or crossreactive for SSeCKS or Gravin. SSeCKS expression was then analysed in developing mouse embryos and in adult tissues. In the foetal mouse, early SSeCKS protein expression (E10–11) is focused in the loose mesenchyme, luminal surface of the neural tube, notochord, early heart and pericardium, urogenital ridge, and dorsal and ventral sections of limb buds. In later stages (E12–14), SSeCKS is widely expressed in mesenchymal cells but is absent in the spinal ganglia. By E15, SSeCKS expression is ubiquitous, although the staining pattern varies from being striated within smooth muscle sarcomeres to filamentous in mesenchymal and select epithelial cells. In the adult mouse, SSeCKS staining is relatively ubiquitous, with highest expression in the gonads, smooth and cardiac muscle, lung, brain and heart. High expression is also detected in fibroblasts and nerve fibres as well as in more specialized cells such as glomerular mesangial cells and testicular Sertoli cells. SSeCKS expression in the rat testes correlates with the induction of puberty, and in mature mouse spermatozoa, SSeCKS is found in peripheral acrosome membranes and in a helix-like winding pattern within the midsection. Periodic enrichments of SSeCKS are found in sperm midsections and in developing axons, suggesting a role in architectural infrastructure. As with Gravin, high SSeCKS expression is absent in most epithelial cells; however, in contrast to Gravin, SSeCKS is expressed in Purkinje cells, cardiac muscle, macrophages and hepatic stellate cells, indicating overlapping yet distinct patterns of tissue expression in the SSeCKS/Gravin family. The data suggest roles for SSeCKS in the control of cytoskeletal and tissue architecture, formation of migratory processes and cell migration during embryogenesis.

Cell-cycle regulated expression and serine phosphorylation of the myristylated protein kinase C substrate, SSeCKS: Correlation with culture confluency, cell cycle phase and serum response

We recently identified a novel myristylated protein kinase C (PKC) substrate, named SSeCKS (pronounced essex), whose transcription is suppressed >15 fold in src- or ras-transformed rodent fibroblasts, but not in raf-transformed cells [1, 2]. SSeCKS associates with and controls the elaboration of a cortical cytoskeletal matrix in response to phorbol esters [2], and overexpression of SSeCKS causes growth arrest of untransformed NIH3T3 cells [3]. Our preliminary data suggested that SSeCKS functions as a negative mitogenic regulator by controlling cytoskeletal architecture and that serine phosphorylation of SSeCKS by kinases such as PKC alters its interaction with cytoskeletal matrices and its ability to control mitogenesis. Here, we determine the effects of culture confluency, growth arrest and serum response on the steady-state abundance of SSeCKS RNA and protein and on the relative level of phosphoserine-free SSeCKS. SSeCKS transcription is initially induced by serum factors and by cont act-inhibited growth rather than by cell-cycle arrest induced by serum starvation, hydroxyurea or nocodazole, and following serum-induced G1/S progression, SSeCKS transcription is suppressed. SSeCKS protein is hyperphosphorylated on serine residues during G1/S progression but not during the G2/M phase. Finally, we show that the induction of SSeCKS protein expression by contact inhibition is independent of SSeCKS serum responsiveness. These data suggest that SSeCKS expression and function can be controlled at either the transcriptional or post-translational level in response to serum factors and culture confluency. The data strengthen the notion that SSeCKS plays an important, yet transient, role in cell cycle progression from G0 to G1 that differs from its role in controlling contact-inhibited growth. (Mol Cell Biochem 175: 233–241, 1997)

HIV-1 expression induces cyclin D1 expression and pRb phosphorylation in infected podocytes: cell-cycle mechanisms contributing to the proliferative phenotype in HIV-associated nephropathy

BackgroundThe aberrant cell-cycle progression of HIV-1-infected kidney cells plays a major role in the pathogenesis of HIV-associated nephropathy, however the mechanisms whereby HIV-1 induces infected glomerular podocytes or infected tubular epithelium to exit quiescence are largely unknown. Here, we ask whether the expression of HIV-1 genes in infected podocytes induces cyclin D1 and phospho-pRb (Ser780) expression, hallmarks of cyclin D1-mediated G1 → S phase progression.ResultsWe assessed cyclin D1 and phospho-pRb (Ser780) expression in two well-characterized models of HIV-associated nephropathy pathogenesis: HIV-1 infection of cultured podocytes and HIV-1 transgenic mice (Tg26). Compared to controls, cultured podocytes expressing HIV-1 genes, and podocytes and tubular epithelium from hyperplastic nephrons in Tg26 kidneys, had increased levels of phospho-pRb (Ser780), a target of active cyclin D1/cyclin-dependent kinase-4/6 known to promote G1 → S phase progression. HIV-1-infected podocytes showed markedly elevated cyclin D1 mRNA and cyclin D1 protein, the latter of which did not down-regulate during cell-cell contact or differentiation, suggesting post-transcriptional stabilization of cyclin D1 protein levels by HIV-1. The selective suppression of HIV-1 transcription by the cyclin-dependent kinase inhibitor, flavopiridol, abrogated cyclin D1 expression, underlying the requirement for HIV-1 encoded products to induce cyclin D1. Indeed, HIV-1 virus deleted of nef failed to induce cyclin D1 mRNA to the level of other single gene mutant viruses.ConclusionsHIV-1 expression induces cyclin D1 and phospho-pRb (Ser780) expression in infected podocytes, suggesting that HIV-1 activates cyclin D1-dependent cell-cycle mechanisms to promote proliferation of infected renal epithelium.

Induction of Apoptosis by HIV-1-Infected Monocytic Cells

We have previously described a soluble 6000-Da peptide produced by an HIV-1-infected human macrophage cell line, clone 43HIV, which induces apoptosis in T and B cells. We have identified this factor as the novel cDNA clone FL14676485 that encodes for the human hypothetical protein, FLJ21908. The FL14676485 cDNA clone was isolated from a 43HIV λ ZAP Escherichia coli expression library and screened with a panel of rabbit and mouse anti-apoptotic Abs. We transfected the FL14676485 clone into Bosc cells and non-HIV-1-infected 43 cells. Western blot analysis of lysates from the FL14676485-transfected 43 cells and Bosc cells using anti-proapoptotic factor Abs revealed a protein with a molecular mass of 66 kDa corresponding to the size of the full-length gene product of the FL14676485 clone, while Western blot of the supernatant demonstrated a doublet of 46-kDa and 6000-Da peptide that corresponds to our previously described proapoptotic factor. Primary HIV-1BaL-infected monocytes also produce the FLJ21908 protein. Supernatants from these transfected cells induced apoptosis in PBMC, CD4+, and CD8+ T and B cells similar to the activity of our previously described proapoptotic factor. PCR analysis of 43 cells and 43HIV cells revealed a base pair fragment of 420 bp corresponding to the FL14676485 gene product in 43HIV cells, but not in 43 cells. The FLJ21908 protein induces apoptosis through activation of caspase-9 and caspase-3. We have further demonstrated that the FLJ21908 protein has apoptotic activity in the SH-SY5Y neuronal cell line and can be detected in brain and lymph tissue from HIV-1-infected patients who have AIDS dementia. The FLJ21908 protein may contribute to the apoptosis and dementia observed in AIDS patients.

Reexpression of the Major PKC Substrate, SSeCKS, Correlates with the Tumor‐Suppressive Effects of SCH51344 on Rat‐6/src and Rat‐6/ras Fibroblasts but Not on Rat‐6/raf Fibroblasts

Oncogenes such as v-src and v-ras induce oncogenic transformation by activating at least two signaling pathways, a Raf-Mek-Erk pathway controlling proliferationinducing transcription, and pathways involving Rho-family GTPases that regulate both proliferation and control of cytoskeletal architecture. SCH51344 is a pyrazoloquinoline derivative that was isolated by its ability to derepress a transformationsensitive α-actin isoform and was subsequently shown to suppress ras-induced oncogenic transformation by inhibiting membrane ruffling controlled by the Rho-family member, Rac.1–3 Because SCH51344 does not inhibit ras activation of either Erk or Jnk, this drug is thought to mediate tumor suppression by reestablishing controls on actin-based cytoskeletal organization rather than by affecting proliferative signaling. We previously described a new cell cycle-regulated protein kinase C (PKC) substrate, SSeCKS, that interacts with and modulates the organization of the actin-based cytoskeleton.4–7 SSeCKS expression is downregulated in srcand ras-, but not in raf-transformed rodent fibroblasts,4 and its reexpression in src-transformed fibroblasts or ras-transformed prostate cancer epithelial cells suppresses anchorageand mitogen-independence and reestablishes contact-inhibited growth and cytoskeletal organization.8 SSeCKS also shares significant similarity with human Gravin, a scaffolder of kinases such as PKC and PKA.9 Therefore, we surmised that SSeCKS expression might serve as a marker for oncogenes that alter the critical cytoskeletal control pathways involved in transformation. Here we ask if SCH51344 could derepress SSeCKS and if this correlates with a loss of anchorage independence in src-, ras-, and raf-transformed rat-6 fibroblasts.