Oncogene (2014) 33, 1167–1180 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE A novel splice variant of calcium and integrin-binding protein 1 mediates protein kinase D2-stimulated tumour growth by regulating angiogenesis M Armacki1,2, G Joodi2, SC Nimmagadda2, L de Kimpe3,4, GV Pusapati5, S Vandoninck4, J Van Lint4, A Illing1 and T Seufferlein1,2 Protein kinase D2 (PKD2) is a member of the PKD family of serine/threonine kinases, a subfamily of the CAMK super-family. PKDs have a critical role in cell motility, migration and invasion of cancer cells. Expression of PKD isoforms is deregulated in various tumours and PKDs, in particular PKD2, have been implicated in the regulation of tumour angiogenesis. In order to further elucidate the role of PKD2 in tumours, we investigated the signalling context of this kinase by performing an extensive substrate screen by in vitro expression cloning (IVEC). We identified a novel splice variant of calcium and integrin-binding protein 1, termed CIB1a, as a potential substrate of PKD2. CIB1 is a widely expressed protein that has been implicated in angiogenesis, cell migration and proliferation, all important hallmarks of cancer, and CIB1a was found to be highly expressed in various cancer cell lines. We identify Ser118 as the major PKD2 phosphorylation site in CIB1a and show that PKD2 interacts with CIB1a via its alanine and proline-rich domain. Furthermore, we confirm that CIB1a is indeed a substrate of PKD2 also in intact cells using a phosphorylation-specific antibody against CIB1a-Ser118. Functional analysis of PKD2-mediated CIB1a phosphorylation revealed that on phosphorylation, CIB1a mediates tumour cell invasion, tumour growth and angiogenesis by mediating PKD-induced vascular endothelial growth factor secretion by the tumour cells. Thus, CIB1a is a novel mediator of PKD2-driven carcinogenesis and a potentially interesting therapeutic target. Oncogene (2014) 33, 1167–1180; doi:10.1038/onc.2013.43; published online 18 March 2013 Keywords: PKD2; CIB1; angiogenesis; carcinogenesis INTRODUCTION expression cloning (IVEC) to identify potential novel PKD The protein kinase D (PKD) family belongs to the calcium/ substrates that could have an impact on angiogenesis. One calmodulin-dependent protein kinase super-family and comprises potentially novel substrate identified in the screen was a splice PKD1, PKD2 and PKD3.1 PKDs are activated either directly via variant of human calcium and integrin-binding protein 1 (CIB1) phorbol esters or indirectly by various mechanisms including G that we termed CIB1a (GenBank accession number: BankIt1498958 protein-coupled receptors.2 These kinases participate in many key Seq1 JQ246073). signalling pathways in a diverse range of cells and are involved in CIB1 is a 22-kDa EF-hand-containing protein identified originally fundamental biological processes: PKDs have a critical role in as a binding partner of the platelet integrin aIIb, and later found to motility, migration and invasion of cancer cells.3–6 PKD isoforms, inhibit aIIbb3 activation in megakaryocytes.16,17 CIB1 is widely in particular PKD2, are highly expressed in certain tumours7,8 expressed18,19 and has a role in angiogenesis. Apparently, it is not and have been implicated in the regulation of tumour cell required for developmental angiogenesis, as CIB1-KO mice are proliferation and apoptosis.9,10 Recently, we and other viable and female mice are fertile, but is essential for proper EC laboratories have demonstrated that PKD1 and PKD2 have signalling and functions such as migration, proliferation and critical roles in vascular biology and angiogenesis.7,11–15 PKD2 is nascent tubule formation.20,21 Loss of CIB1 in ECs results in a pivotal mediator of vascular endothelial growth factor (VEGF)- attenuated responses to angiogenic growth factors such as VEGF induced endothelial cell (EC) proliferation and migration and and fibroblast growth factor 2 (FGF2), and consequently regulates the expression of VEGF receptor-2 and the production decreased expression of the matrix-degrading proteinase matrix of cytokines in ECs.13 Furthermore, PKD2 has a major role in metalloproteinase 2. CIB1 expression in the host also promotes tumour angiogenesis by mediating hypoxia-induced VEGF tumour growth and tumour-induced angiogenesis by an as yet production and secretion in tumour cells, as well as VEGF- unknown mechanism.22 CIB1 physically associates with proteins induced signalling in the tumour-associated ECs.7 PKD2 such as the transcription factor PAX3,23 the inositol 1, 4, downstream targets responsible for its effect on angiogenesis 5-trisphosphate receptor,24 polo-like kinases,18,25 Rac 3,26 focal are as yet incompletely understood. adhesion kinase (FAK)27 and p21-activated kinase (PAK1).28 For a better understanding of the precise signalling context of Among these binding partners, PAK1 and FAK regulate EC PKD2, in particular with respect to angiogenesis, we used in vitro function and angiogenesis in vitro or in vivo.29–31 1Department of Internal Medicine I, University of Ulm, Ulm, Baden-Wuerttemberg, Germany; 2Department of Internal Medicine I, Martin-Luther-University Halle-Wittenberg, Halle(Saale), Germany; 3Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands; 4Department of Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven, Belgium and 5Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA. Correspondence: Professor T Seufferlein, Department of Internal Medicine I, Ulm University, Albert-Einstein-Allee 23 D-89081 Ulm-89081, Baden-Wuerttemberg, Germany. E-mail: [email protected] Received 10 January 2012; revised 8 January 2013; accepted 11 January 2013; published online 18 March 2013 CIB1a mediates PKD2-induced tumour growth and angiogenesis M Armacki et al 1168 Here we identify a novel splice variant of CIB1, termed CIB1a, as been implicated in angiogenesis, we decided to further examine a substrate of PKD2 both in vitro and in intact cells, and found this clone, which we termed CIB1a. CIB1a is generated by an Ser118 as the major PKD2 phosphorylation site in CIB1a. Our data intron retaining mode of splicing and has an additional 120 bp also show that CIB1a specifically interacts with the N-terminal located in the coding region of N-terminal part of cDNA. The alanine and proline (AP)-rich domain of PKD2. CIB1a phosphoryla- retained part of the intron encodes amino acids properly, tion by PKD2 results in increased VEGF-A secretion by the containing no stop codon and causing no shift in the reading tumour cells in vitro and stimulates tumour growth and tumour frame (Supplementary Figure S1). angiogenesis on the chicken chorioallantois and enhances tumour To confirm that CIB1a is a direct substrate of PKD2, we cell invasion, making CIB1a a novel downstream mediator of the generated recombinant CIB1a protein from bacteria. Phosphoryla- pro-angiogenic properties of PKD2 in cancer. tion of CIB1a by PKD2 was analysed by in vitro kinase (IVK) assays. Wild-type PKD2 (PKD2-WT) phosphorylated CIB1a in vitro. CIB1a phosphorylation was substantially enhanced when PKD2 was RESULTS activated by phorbol 12-myristate 13-acetate (PMA) or on Identification of potential PKD2 substrates involved in incubation with constitutively active PKD2 (PKD2-2SE). In contrast, angiogenesis CIB1a phosphorylation was virtually undetectable in the presence To identify potential novel PKD2 substrates that could mediate the of catalytically inactive PKD2 (PKD2-DA; Figure 1b). Thus, PKD2 effect of PKD2 in tumour angiogenesis, we performed IVEC.32 phosphorylates CIB1a in vitro. Pools of approximately 100 clones from a complementary DNA CIB1 is widely expressed in normal human tissues.19 (cDNA) library were transcribed and translated in vitro, and the Interestingly, high levels of CIB1 expression were observed in resulting S35-labelled protein pools were incubated with or breast cancer cell lines and tissues18 as well as in bladder, cervix without catalytically active PKD2 (PKD2-2SE) and subjected to and colorectal cancer using microarrays and serial analysis of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– gene expression (SAGE) (GeneNote, http://bioinfo2.weizmann.ac.il; PAGE). Phosphorylation of potential substrates was assessed by GeneCards; http://www.genecards.org). To determine the the kinase-dependent alteration in electrophoretic mobility expression of CIB1 and CIB1a in various human cancer cell lines, (Figure 1a, upper panel). Positive cDNA pools were progressively we analysed their expression by reverse transcriptase–PCR using subdivided and single cDNA sub-pools were re-assayed for the primers designed to amplify both a 0.4-kb fragment of CIB1 and a presence of a kinase-induced mobility shift (Figure 1a, lower 0.5-kb fragment of CIB1a (Figure 1c). We detected a single 0.4-kb panel). This procedure yielded specific cDNAs encoding putative band in all cell lines examined (Figure 1c, upper panel), indicating PKD2 substrates. One positive clone with an open reading frame the presence of CIB1-specific mRNA. We speculated that the CIB1a of 697 bp encoding a polypeptide with a predicted mass of 26 kDa isoform might have also been amplified, but this could not be corresponded to a splice variant of the human CIB1. As CIB1 has demonstrated in the same PCR reaction. Therefore, CIB1a was re- protein pools screen kDa IVK 32 clone 2 26 WB:GFP PKD2 130 PKD2-WT ++---- PKD2-DA --- ++ - PKD2-2SE - --- + + PMA - + - + - + PKD2-2SE-++ -+-- + single protein screen clone 2 mobility shift Myc-CIB1 + - PKD2-2SE - + Myc-CIB1a - + kDa32 CIB1a (ectopic) 26 CIB1 WCL WB:CIB AGSe HeLa Cos1 MiaPaca1Panc1 HEK293U87 CIB1 HeLa HEK293U87 Panc1 AGSe MiaPaca1 kDa32 CIB1a CIB1a 26 (endo) RT-PCR WCL WB:CIB Figure 1. (a) Identification of PKD2 substrate/s via IVEC. (a, upper panel) 35S-labelled protein pools were generated by in vitro transcription and translation of a plasmid based, human adult brain cDNA library, using Promega’s Gold TnT SP6 Express 96 coupled Transcription and Translation System and incubated with ( þ ) or without ( À ) catalytically active PKD2 (PKD2-2SE (S706/710E)).