A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea.

The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation.

Lysophosphatidic acid rescues human dental pulp cells from ischemia-induced apoptosis.

INTRODUCTION: Dental pulp is particularly susceptible to ischemic conditions (hypoxia and serum deprived) because it is commonly exposed to trauma, inflammation, chronic caries injury, and pulpitis. We investigated the apoptotic response of human dental pulp cells (HDPCs) to varying levels of oxygen and serum to mimic different degrees of ischemia, tested whether lysophosphatidic acid (LPA) could reverse ischemia-induced apoptosis, and investigated the possible mechanisms of LPA. METHODS: HDPCs were cultured under conditions mimicking serum deprivation and ischemia for 2 days with or without LPA at 25 µg/mL. Flow cytometry and JC-1 fluorescence were used to detect any apoptotic change. Western blotting was used to measure the expression of the apoptosis regulators B-cell lymphoma 2 (Bcl-2) and Bax, focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK), and Akt. RESULTS: Flow cytometry and JC-1 immunofluorescence showed that ischemia could induce apoptosis of HDPCs in 2 days and treatment with LPA could reduce cell death significantly. To clarify the molecular mechanisms, Western blot results showed up-regulation of both proapoptotic Bax and antiapoptotic Bcl-2 during apoptosis. LPA functioned as an antiapoptotic cytokine by activation of the phosphorylation of FAK and ERK. No statistically significant difference was found in the activation levels of p-Src or p-Akt. CONCLUSIONS: A self-defense mechanism functioned during cell apoptosis. LPA could effectively rescue HDPCs from ischemia-induced apoptosis via regulation of Bax and Bcl-2 and the activation of phosphorylated FAK and phosphorylated ERK. LPA is a potent candidate for biological therapy of chronic pulpal inflammatory diseases.

COX-2, TFF1, and Src define better prognosis in young patients with gastric cancer.

BACKGROUND AND OBJECTIVES: Despite its dwindling occurrence, gastric cancer remains a leading cause of cancer related mortality worldwide. Molecular determinants of prognosis that impact survival are being sought out as a means to facilitate rational clinical decision-making and enhance patient management. In this study, we evaluated three molecules implicated in gastric carcinogenesis and demonstrated that the differential expression of cyclooxygenase-2 (COX-2) and the viral oncogene homolog Src proteins could explain the differences in survival observed in patients older and younger than 50 years of age. METHODS: We evaluated 5-year survival in a cohort of 423 gastric cancer patients using chronological age as a variable. Additionally, we assessed the protein expression of three molecules (COX-2, TFF1, Src) implicated in the pathogenesis of gastric cancer using immunohistochemistry. RESULTS: We found that patients younger than 50 years of age had a better 5-year survival rate in all tumor stages. We found that the expression of COX-2 and Src correlated significantly with survival in this group without any significant impact attributable to TFF1. CONCLUSIONS: Our study demonstrates that young gastric cancer patients have a better prognostic outlook that could in part be explained by the differential expression of COX-2 and Src.

Saturated fatty acids induce c-Src clustering within membrane subdomains, leading to JNK activation.

Saturated fatty acids (FA) exert adverse health effects and are more likely to cause insulin resistance and type 2 diabetes than unsaturated FA, some of which exert protective and beneficial effects. Saturated FA, but not unsaturated FA, activate Jun N-terminal kinase (JNK), which has been linked to obesity and insulin resistance in mice and humans. However, it is unknown how saturated and unsaturated FA are discriminated. We now demonstrate that saturated FA activate JNK and inhibit insulin signaling through c-Src activation. FA alter the membrane distribution of c-Src, causing it to partition into intracellular membrane subdomains, where it likely becomes activated. Conversely, unsaturated FA with known beneficial effects on glucose metabolism prevent c-Src membrane partitioning and activation, which are dependent on its myristoylation, and block JNK activation. Consumption of a diabetogenic high-fat diet causes the partitioning and activation of c-Src within detergent insoluble membrane subdomains of murine adipocytes.

Roles of Src-like adaptor protein 2 (SLAP-2) in GPVI-mediated platelet activation SLAP-2 and GPVI signaling.

BACKGROUND: Glycoprotein VI (GPVI) /Fc receptor gamma (FcRγ)-chain complex is one of the collagen receptors in platelets and responsible for the majority of the intracellular signaling events through a similar pathway to immune receptors. Src-like adaptor protein 2 (SLAP-2) is a recently characterized adaptor protein predominantly expressed in hematopoietic cells. In T cells, SLAP-2 was reported to associate with several tyrosine phosphorylated proteins, and function as a negative regulator of signaling downstream of T cell antigen receptor by virtue of its interaction with the ubiquitin ligase c-Cbl. But the data regarding the presence and role of SLAP-2 proteins in platelets is limited. OBJECTIVES: We describe the characterization of SLAP-2 in human platelets. METHODS: Human platelets were analyzed by Western blot analysis, immunoprecipitation, and pull down assay, etc. RESULTS: Immunoprecipitation revealed the presence of two forms of SLAP-2 with approximately 28 kD and 25 kD, and following stimulation of GPVI, the additional form with approximately 32 kD apppeared. We have found that upon GPVI activation, SLAP-2 translocated from the Triton X-100-soluble fraction to the Triton X-100-insoluble cytoskeleton fraction, with concomitant association with Syk, c-Cbl, and LAT. CONCLUSIONS: SLAP-2 appears to play a role in regulating signaling pathways by bringing important signaling molecules such as c-Cbl and Syk into proximity of cytoskeletal substrates. In platelets, SLAP-2 may have function as a negative regulator of GPVI-mediated signaling by interacting with c-Cbl, being similar to that reported in T cells.

Increased levels of active c-Src distinguish invasive from in situ lobular lesions.

INTRODUCTION: Mounting molecular evidence suggests that invasive lobular carcinoma (ILC) is developing from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). However, little is known about the mechanisms promoting the progression of lobular breast cancer (LBC) to invasive disease. Here, we investigated whether c-Src kinase, an established inducer of invasive states, contributes to the progression from ALH/LCIS to ILC. METHODS: Immunochemistry for c-Src and other cancer-related molecules was performed on archived tissue specimens from 57 LBC patients. Relative c-Src activity was estimated by comparing fluorescence intensity of ILC with that of adjacent ALH/LCIS and nonneoplastic epithelia after staining with an antibody against active c-Src. Expression of active c-Src was correlated with markers of invasion and malignancy and with relapse among LBC patients. RESULTS: Levels of activated c-Src were increased in ILC relative to ALH/LCIS (1.63-fold +/- 0.24 SD) and nonneoplastic epithelia (1.47 +/- 0.18 SD). Increased c-Src levels correlated with the activation of c-Src downstream targets (Fak, Stat-3) and the expression of mesenchymal markers. ILC cells with activated c-Src co-expressed metastatic markers (Opn, Cxcr4) and included cells positive for the cancer stem cell marker Aldh1. A tendency for high c-Src levels (P = 0.072) was observed among the seven LBC patients with relapsed disease. CONCLUSIONS: Our data indicate elevated c-Src activity in ILC relative to noninvasive neoplastic tissue. The associated molecular changes suggest that c-Src promotes LBC invasiveness by inducing an epithelial-mesenchymal transition. Therefore, c-Src antagonists might counteract the acquisition of invasiveness during LBC progression. Inhibition of c-Src may also affect ILC cells thought to have a high metastatic potential and to be capable of initiating/maintaining tumor growth. Together with the possible association between high c-Src levels and disease recurrence, our findings encourage the evaluation of c-Src antagonists for the treatment of LBC.

Decreased CHK protein levels are associated with Src activation in colon cancer cells.

Src activation has been associated with colon cancers but the mechanism underlying Src activation is largely unknown. Csk-homologous kinase (CHK) can inhibit the kinase activity of certain Src kinase family members in vitro by phosphorylating the C-terminal tyrosine and by a non-catalytic mechanism. CHK was previously reported to be expressed primarily in brain and hematopoietic cells. We report herein that CHK is also expressed in normal colon cell lines. Furthermore, CHK protein levels are significantly decreased in various colon cancer cell lines and the decrease correlates with the increased specific activity of Src in these cell lines, while the level of the other Src inhibitory kinase, C-terminal Src kinase, is not significantly changed. CHK is also expressed in normal colon tissues but its expression level is decreased in colon cancer tissues collected from the same patients. Immunofluorescence microscopy shows that CHK colocalizes with Src in normal colon FHC cells. Overexpression of CHK in colon cancer cells results in inactivation of Src without phosphorylating Y530 at its C-terminus. In addition, CHK suppresses anchorage-independent cell growth and cell invasion of colon cancer cells. These results reveal a potentially important role for CHK in Src activation and tumorigenicity in colon cancer cells.

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus.

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to approximately 200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.

Expression of c-Src and comparison of cytologic features in cherubism, central giant cell granuloma and giant cell tumors.

Cherubism (CBM) and central giant cell granuloma (CGCG) of the jaw and giant cell tumor (GCT) of the long bone are clinically different diseases. Histologically, they are all multinucleated giant cell (MGC)-containing lesions. This study aims to evaluate the expression of c-Src and cytologic features in CBM, CGCG and GCT and to clarify whether there is a common mechanism underlying the formation of multi-nucleated giant cells (MGCs) in these lesions. Specimens and paraffin blocks were collected from patients with CBM (12 cases), CGCG (24 cases) and GCT (37 cases). Histomorpho-metric differences in MGCs were compared among the three types of lesions. The expression of c-Src by immunohistochemistry and in situ hybridization and the expression of TRAP by enzyme histochemical staining were examined. Expression of c-Src mRNA and protein, as well as TRAP staining, was detected in both MGCs and a fraction of mononuclear cells in all investigated lesions. There are no quantitative differences for cytologic features and c-Src expression among the lesions. The results suggested that CBM, CGCG and GCT have overlapping cytological features at the histological level, and c-Src may be involved in the formation of MGCs in the three different diseases.

Postnatal changes in the expression of p60c-Src in mouse testes.

Src family non-receptor tyrosine kinases are involved in signaling pathways which mediate cell growth, differentiation, transformation and tissue remodeling in various organs. In an effort to elucidate functional involvement of p60c-Src (c-Src) in spermatogenesis, the postnatal changes in c-src mRNA and c-Src protein together with kinase activity and subcellular localization were examined in mouse testes. c-src mRNA levels in testes increased during the first 2 weeks of postnatal development (PND). Following a decrease at puberty (PND 28), the c-src mRNA levels re-increased at adulthood (PND 50). Src kinase activity of testes was low at PND 7 but sharply increased prepubertally (PND 15) and highest at adulthood. Upon Western blotting, the level of c-Src protein was the highest in prepubertal testes but rather decreased in adult testes at PND 50. In adult testes, ubiquitination of c-Src proteins was apparent compared with immature one at PND 7, suggesting active turnover of c-Src by ubiquitination. In immature testes, c-Src immunoreactivity was largely found in the cytoplasm of the Sertoli cells. By contrast, in pubertal and adult testes intense immunoreactivity was localized at the adluminal and basal cytoplasm of Sertoli cells bearing elongated spermatids and early germ cells, respectively. The immunoreactivity of c-Src in the Leydig cells was increased during pubertal development, suggesting the functional involvement of c-Src in differentiated adult Leydig cells. Throughout postnatal development, some spermatogonia and spermatocytes showed intensive c-Src immunoreactivity compared with other germ cells, suggesting a possible role of c-Src in germ cell death. Taken together, it is suggested that c-Src may participate in the remodeling of the seminiferous epithelia and functional differentiation of Leydig cells during the postnatal development of mouse testes.

[Effect of PP60c-Src on angiotensin II-induced signal transduction in rat vascular smooth muscle cells].

OBJECTIVE: To further clarify the mechanism of Ang II-induced intracellular signal transduction in vascular smooth muscle cells(VSMCs) proliferation by observing the effect of c-Src on Ang II-mediated MAPK activation and c-fos protein expressions in rat VSMCs. METHODS: Aortic VSMCs from SD rats were cultured primarily and subcultured, which were transfected with anti-sense c-Src oligodeoxynucleotides(ODNs) wrapped with lipofectin to inhibit c-Src activity and protein production. Untransfected VSMCs were used as control, to observe the role of Ang II stimulation in MAPK activation and c-fos protein expression in VSMC. Protein immunoprecipitation and kinase phosphorylation were employed to measure c-Src kinase activity; MAPK kinase activity was assessed by the phosphorylation rate of the substrate MBP(Myelin Basic Protein); Western blot was used to assess the protein expression of c-Src and c-fos. RESULTS: c-Src protein expressions in VSMC, which were transfected with different concentrations of anti-sense c-Src ODNs, were significantly decreased in a negative dose-effect manner (0.2 microm, 0.5 microm, 1.0 microm and 2.0 microm were 68.2%, 34.7%, 30.3% and 15.8% respectively compared with control). c-Src kinase activity was also obviously inhibited. Following stimulation of Ang II on VSMC transfected with anti-sense c-Src ODNs, the increase of c-Src activity was only 8.7% of control,the activity of MAPK only 1.6% compared with control, and the increase in c-fos protein expression 30.3% as control. CONCLUSION: Ang II can induce c-Src activation and intracellular signal transduction in VSMC which depend on c-Src activation, indicating that c-Src is a pivotal signal factor in VSMC proliferation.

Neutral loss analysis in MALDI-MS using an ion-gate scanning mode.

Matrix-assisted laser desorption/ionization reflector time-of-flight (MALDI-reTOF) and electrospray ionization (ESI) mass spectrometry (MS) have become essential tools for the characterization of peptides and proteins. Whereas ESI in combination with a triple quadrupole analyzer allows product ion, precursor ion, and neutral loss analyses, MALDI-reTOF instruments can only be used to record product ion spectra based on the in-source or postsource decay (PSD). We describe a new method to perform neutral loss analyses in MALDI-reTOF instruments in a manner that identifies posttranslationally modified peptides and furthermore retrieves sequence information from peptides. The method is based on the selection of ions in a small time interval to record only signals within the corresponding mass interval. By stepping the time interval through the complete mass range, we obtained a spectrum of stable ions by combining the signals of all individually recorded time intervals. This method furthermore permits PSD fragment ions to be identified, since they reach the detector earlier than the stable ions transmitted in the chosen time interval. The neutral loss analysis were calculated by correlating the PSD fragment ions to the corresponding parent ion detected in this time interval. Moreover, this MALDI-MS mode increased the number of detectable signals in complex peptide mixtures and the signal-to-noise ratio.

Regulation of pp60(c-src) synthesis in rat hippocampal slices by in vitro ischemia and glucocorticoid administration.

Corticosteroids, released from the adrenal gland in response to stress, bind to receptors that act as transcription factors to alter gene expression and, subsequently, protein synthesis. Using [(35)S]-methionine-cysteine incorporation to measure protein synthesis in hippocampal slices incubated under ischemic conditions, synthesis of 60 kDa and 78 kDa proteins decreases 4 hr after in vivo administration of corticosterone to rats. The former protein has been identified by immunoblotting and immunoprecipitation to be the proto-oncogene, pp60(c-src). In the absence of prior glucocorticoid administration, ischemic conditions increase the amount of immunoreactive pp60(c-src) in membranes of hippocampal slices. Chronic exposure to elevated titers of glucocorticoids has been demonstrated to result in cell loss as well as in reduced neuronal plasticity and regeneration. Given the involvement of pp60(c-src) in synaptic plasticity and cell growth, glucocorticoid-mediated reduction in its synthesis is a potential molecular marker for stress-induced alterations in brain function.

GP2, a GPI-anchored protein in the apical plasma membrane of the pancreatic acinar cell, co-immunoprecipitates with src kinases and caveolin.

We previously showed that endocytosis at the apical plasma membrane (APM) of the pancreatic acinar cell is activated by the cleavage of GP2, a GPI-linked protein, from the apical cell surface. This endocytic process, as measured by horseradish peroxidase uptake into pancreatic acinar cells, is blocked by the tyrosine kinase inhibitors genistein and tyrphostin B42 as well as by disruption of actin filaments with cytochalasin. This suggests that the cleavage of GP2 from the cell membrane may activate endocytosis through a tyrosine kinase-regulated pathway. However, the mechanism by which GP2 and tyrosine kinases act together to activate endocytosis at the APM remains unknown. In this study, we demonstrate that pp60, p62yes, caveolin, and annexin, which have previously been implicated in endocytosis in other cell lines, were present in high abundance in GPI-enriched membranes by Western blot analysis. pp60, p62yes, and caveolin all co-immunoprecipitated with GP2 except annexin. An 85-kDa protein whose tyrosine-dependent phosphorylation is correlated with the activation of endocytosis in intact acinar cells also was present in these immunoprecipitates. This suggests that in pancreatic acini, GP2 may exist in a complex with src kinases, caveolin, and an 85-kDa phosphorylated substrate to regulate endocytosis at the APM.

Association of renal cell carcinoma antigen, disialylgalactosylgloboside, with c-Src and Rho A in clustered domains at the surface membrane.

Disialylgalactosylgloboside (DSGG), defined by monoclonal antibody RM2, is a renal cell carcinoma (RCC)-associated antigen which mediates adhesion of RCC TOS-1 cells to certain lung tissue target cells. This adhesion process may initiate preferential lung metastasis of RCC. Ganglioside GM3 is a B16 melanoma-associated antigen which similarly adheres to target cells and promotes consequent metastasis. In view of the close association of GM3-enriched microdomain with transducer molecules c-Src, Rho A, and FAK in B16 cells, we investigated the organizational status of DSGG in RCC cell line TOS-1, with the following results: i) DSGG, but not monosialylgalactosylgloboside, showed extensive clustering at the TOS-1 cell surface; ii) a low-density membrane fraction isolated from TOS-1 cells contained >95% of cellular DSGG, although protein content in this fraction was <1% of total cellular protein; iii) this fraction contained c-Src, Rho A, and FAK, but not H-Ras; iv) c-Src and Rho A were co-immunoprecipitated with DSGG through anti-DSGG mAb RM2 (IgM) affixed to a column. These observations indicate that DSGG is clustered in RCC, as typified by TOS-1 cells, to form a microdomain in which it is closely associated with c-Src, Rho A, and FAK, and may constitute a functional unit as has been observed for GM3 with transducer molecules in B16 cells. The functional organization of such units may be essential in determining malignant properties of RCC cells.

Vascular endothelial growth factor induces activation and subcellular translocation of focal adhesion kinase (p125FAK) in cultured rat cardiac myocytes.

Vascular endothelial growth factor (VEGF) has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation. Recently, we found that VEGF activated the mitogen-activated protein kinase cascade in cultured rat cardiac myocytes. To elucidate how VEGF affects adhesive interaction of cardiac myocytes with the extracellular matrix (ECM), one of the important cell functions, we investigated the molecular mechanism of activation of focal adhesion-related proteins, especially focal adhesion kinase (p125(FAK)), in cultured rat cardiac myocytes. We found that the 2 VEGF receptors, KDR/Flk-1 and Flt-1, were expressed in cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation. VEGF induced tyrosine phosphorylation and activation of p125(FAK) as well as tyrosine phosphorylation of paxillin; this was accompanied by subcellular translocation of p125(FAK) from perinuclear sites to the focal adhesions. This VEGF-induced activation of p125(FAK) was inhibited partially by the tyrosine kinase inhibitors genistein and tyrphostin. Activation of p125(FAK) was accompanied by its increased association with adapter proteins GRB2, Shc, and nonreceptor type tyrosine kinase p60(c-src). Furthermore, we confirmed that VEGF induced a significant increase in adhesive interaction between cardiac myocytes and ECM using an electric cell-substrate impedance sensor. These results strongly suggest that p125(FAK) is one of the most important components in VEGF-induced signaling in cardiac myocytes, playing a critical role in adhesive interaction between cardiac myocytes and ECM.

Overexpression and activation of the tyrosine kinase Src in human pancreatic carcinoma.

Src family tyrosine kinases participate in the regulation of cell adhesion, cell growth and differentiation. Here, we examine for the first time the potential role of Src for growth regulation of human pancreatic carcinoma cells. By immunohistochemical analysis, Src was overexpressed in 13/13 pancreatic carcinoma tissue but not in 6 normal pancreatic tissue specimen. In Western blots of total cellular extracts, Src protein expression was elevated in 14/17 carcinoma cell lines as compared to normal pancreas or cultured human pancreatic duct cells. Kinase activity was only detectable in cancer cells and did not correlate with the amount of kinase protein or with the expression of the regulatory kinase Csk, indicating that Src is not regulated through protein expression or through expression of Csk. The Src-specific tyrosine kinase inhibitor herbimycin A decreased cell growth in a dose-dependent manner. We suggest that Src family kinases participate in growth regulation of pancreatic cancer cells.

Substrate recognition by osteoclast precursors induces C-src/microtubule association.

The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-src) (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.