We have Identified the nucleotide sequence of the cDNA encoding the human counterpart of the moue gene Plk (polo-like kinase). . Activated Ras mediates signal transmission from receptor tyrosine kinases to a cascade of serine/threonine kinases including c-Raf, MEK, MAP kinase, and RSK (9). Many observations suggest that MAP kinase and RSK, which are located in the cytoplasm and in the nucleus, directly influence gene expression by phosphorylation of transcription factors (10, 11). Very recently, a MEK kinase independent ofRaf-1 has been described (12). Still, many components of this mitogenic cascade, such as protein kinases and many of their interactions, remain to be discovered.We and others have cloned cDNAs of novel protein kinases by using degenerate oligonucleotide primers for the PCR amplification of reverse-transcribed mRNA (13)(14)(15)(16)(17). A principal attraction of this approach is that based on conserved motifs of the kinase domain it allows amplification of protein kinases which are expressed at very low frequency. Here, we describe the PCR-based identification of a gene coding for a protein kinase from embryonic tissue. The complete cDNA was subsequently isolated from a cDNA library based on RNA from a squamous-cell lung carcinoma. The cDNA encodes a protein which seems to be the human counterpart of a mouse protein referred to as Plk (polo-like kinase) and contains structural hallmarks of protein-serine/ threonine kinases.t We show that expression of human PLK mRNA is increased in proliferating tissues like human tumors, as well as in cell lines and growth-stimulated primary cells. In quiescent cells, PLK transcripts were not found. These data suggest that PLK mRNA expression is tightly linked to proliferation.

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequencespecific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.The effects of many growth factors and cytokines are mediated by high-affinity binding to receptor tyrosine kinases (RTKs) resulting in autophosphorylation of the cytoplasmic domain (1). Phosphotyrosine residues serve as binding sites for downstream signaling proteins that establish a complex network of interactions within the cell. Modular structures involved in binding of activated RTKs identified so far include Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains, which both bind specifically to phosphorylated tyrosine residues in a sequencespecific fashion (2).Recently, we isolated two Eph-related RTKs, named EphB3 and EphB2 (refs. 3 and 4; B.B., U.H., T.K., and K.S., unpublished data), which both bind specifically to the transmembrane subgroup of Eph-receptor ligands (5, 6). The genetic analysis of EphB2 and EphB3 revealed a physiological requirement of both receptors for pathfinding of specific commissural axons in the central nervous system (7,8). To date, the information about signaling molecules mediating Eph-receptor-specific responses is still limited to a few family members. Interactions have been described for the activated Eph-family members EphA2, EphA4, and EphB1 and the SH2-domain-containing proteins p85 subunit of phosphatidylinositol 3-kinase, the adapter protein SLAP, Grb2, Grb10, and Fyn (8-11). Lately, we found that activated EphB3 interacts with Crk, Fyn, and rasGAP in a SH2-dependent manner (12): Autophosphorylation of Tyr-614 in the juxtamembrane region of the receptor generates a multi-docking-site for these interactions...

Signal transduction from the extracellular environment across the cell membrane governs cellular growth and differentiation. These signals are often transduced by receptor tyrosine kinases (RTKs) 1 (1, 2). Defects in RTK genes, their overexpression, or autocrine ligand production may play a critical role in the induction and progression of certain neoplasms (3).Based on structural considerations RTKs have been divided into 14 sub-families (2). One of these is the EPH family of receptors, which constitutes the largest known family of RTKs, named for the prototype member EPH of this family (2). To date, cDNA sequences for nine distinct human EPH family members have been reported: EPH (4), ECK (5), HEK (6), HEK2 (7), HTK (8), ERK (9, 10) and HEK7,. Despite the large number of RTKs in the EPH family, all of the molecules were identified as orphan receptors without known ligands. This situation has hindered understanding the biochemical roles of this family of RTKs.The first ligand to be identified was B61 (12, 13). B61, a tumor necrosis factor ␣-induced gene product from cultured human umbilical vein endothelial cells was found to bind the EPH receptor family member ECK. We have also found that B61 is a ligand for HEK and elk and have named it LERK-1 (14). Subsequently, six additional ligands for the EPH family of RTKs were identified which we have named LERK-2 through LERK-7 (14 -18). These ligands have an amino acid sequence identity ranging between 27 and 59% and are membranebound. The LERKs can be subdivided into two groups based on their mechanism of membrane attachment. LERK-1, LERK-3, LERK-4, LERK-6, and LERK-7 are anchored by glycosylphosphatidylinositol linkage while LERK-2 and LERK-5 are type 1 transmembrane proteins.Here we describe that within the group of LERK proteins, LERK-2 and LERK-5 bind to HEK2 expressed in 32D-myeloid progenitor cell line, whereas LERK-1, LERK-3, LERK-4, and LERK-7 do not. In addition, soluble forms of LERK-2 stimulate autophosphorylation of HEK2 so that membrane attachment does not seem to be required for activation of the HEK2 receptor kinase. Furthermore, we provide evidence for stable cell-cell contacts mediated by the interaction of LERK-2 and HEK2 expressing cells and subsequent juxtacrine stimulation of receptor autophosphorylation. MATERIALS AND METHODSReagents-Rabbit polyclonal antisera to HEK2 were generated by immunizing animals with a peptide corresponding to amino acid residues 897-998 (7). Monoclonal anti-phosphotyrosine antibodies PY20 were purchased from Transduction Laboratories. Sepharose-protein A beads, horseradish peroxidase-conjugated goat anti-mouse, and goat anti-rabbit antibodies were from Sigma. The mammalian expression vectors pRc/CMV and pCEP4 were supplied by Invitrogen. Gentamicin sulfate (G418) was obtained from Life Technologies, Inc., hygromycin B was from Boehringer Mannheim. Generation of a Kinase-negative Mutant of HEK2 and Construction of Expression Vectors with HEK2 Receptors or LERK-2-The HEK2cDNA, containing the complete open reading frame ...

HEK2 belongs to the family of EPH-related receptor tyrosine kinases (RTK) which are involved in axonal path®nding and the formation of the embryonic body plan. The knowledge about intracellular pathways of signal transduction mediated by EPH-related receptors is still limited. Many of the known key players of cellular signalling contain Src homology 2 (SH2) domains, which recognize phosphotyrosine motifs in RTKs. Thus, we examined the interactions of various SH2-containing molecules like PLC-g1, rasGAP, p85 subunit of PI3-kinase, Src, Fyn, Crk, Nck, Grb2 and Shc with HEK2 using in vitro binding assays, immunoprecipitations and yeast Two-Hybrid assays. We found that rasGAP, Crk and Fyn bind in a SH2-dependent manner to autophosphorylated HEK2. rasGAP, which contains two SH2-and one SH3-domain, was shown to associate with its Nterminal SH2-domain to HEK2. Furthermore, we demonstrated that a single amino acid substitution (Y614F) clearly reduces the phosphotyrosine content of HEK2 and abrogates its ability to bind rasGAP, Crk and Fyn indicating that this residue functions as major phosphorylation and multi-docking site. The conservation of this predicted binding site among various EPH-related RTKs provides evidence that Fyn, Crk and rasGAP are key players in signal transduction of at least a subset of these receptors.

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BackgroundOver the past two decades, there has been a rising trend in malignant melanoma incidence worldwide. In 2008, Germany introduced a nationwide skin cancer screening program starting at age 35. The aims of this study were to analyse the distribution of malignant melanoma tumour stages over time, as well as demographic and regional differences in stage distribution and survival of melanoma patients.MethodsPooled data from 61 895 malignant melanoma patients diagnosed between 2002 and 2011 and documented in 28 German population-based and hospital-based clinical cancer registries were analysed using descriptive methods, joinpoint regression, logistic regression and relative survival.ResultsThe number of annually documented cases increased by 53.2% between 2002 (N = 4 779) and 2011 (N = 7 320). There was a statistically significant continuous positive trend in the proportion of stage UICC I cases diagnosed between 2002 and 2011, compared to a negative trend for stage UICC II. No trends were found for stages UICC III and IV respectively. Age (OR 0.97, 95% CI 0.97–0.97), sex (OR 1.18, 95% CI 1.11–1.25), date of diagnosis (OR 1.05, 95% CI 1.04–1.06), ‘diagnosis during screening’ (OR 3.24, 95% CI 2.50–4.19) and place of residence (OR 1.23, 95% CI 1.16–1.30) had a statistically significant influence on the tumour stage at diagnosis. The overall 5-year relative survival for invasive cases was 83.4% (95% CI 82.8–83.9%).ConclusionsNo distinct changes in the distribution of malignant melanoma tumour stages among those aged 35 and older were seen that could be directly attributed to the introduction of skin cancer screening in 2008.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2963-0) contains supplementary material, which is available to authorized users.

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