SRPK1 and LBR Protein Kinases Show Identical Substrate Specificities Stamatia Papoutsopoulou, Eleni Nikolakaki, and Thomas Giannakouros Laboratory of Biochemistry, School of Chemistry, Aristotelian University of Thessaloniki, Thessaloniki 54 006, Greece Received January 19, 1999 Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing and the lamin B receptor (LBR), an integral protein of the inner nuclear mem- brane, are among the best characterized proteins that contain RS domains. Two SR Protein-specific Kinases, SRPK1 and SRPK2, have been shown to phosphorylate specifically the RS motifs of the SR family of splicing factors and play an important role in regulating both the spliceosome assembly and their intranuclear dis- tribution, whereas an LBR-associated kinase, that spe- cifically phosphorylates a strech of RS repeats located at the NH 2 -terminal region of LBR, has been recently purified and characterized from turkey erythrocyte nuclear envelopes. Using synthetic peptides repre- senting different regions of LBR and recombinant pro- teins produced in bacteria we now demonstrate that SRPK1 modifies LBR with similar kinetics and on the same sites as the LBR kinase, that are also phosphor- ylated in vivo. These data provide significant evidence for a new role of SRPK1 in addition to that of pre- mRNA splicing. © 1999 Academic Press Key Words: SRPK1; LBR kinase; SR proteins; LBR. RS protein kinases constitute a novel class of en- zymes that specifically modify arginine/serine dipep- tide motifs. Several proteins were found to contain RS domains that differ in length, number of arginine/ serine dipeptides and content of other amino acids (for relevant information see Ref. 1 and and an automatic update of RS domain-containing proteins under the following electronic address http://www.mann.embl- heidelberg.de/Services/PeptideSearch/PeptideSearch Intro.html). A variety of in vitro and in vivo techniques including co-precipitation, yeast two-hybrid assays, and far-western blots have revealed that RS regions can mediate protein-protein interactions (for review see Ref. 2). A number of RS domains were also found to infuence RNA binding (3), promote RNA-RNA anneal- ing (4) or contain sequences that act as subcellular localization signals (5). Among the best characterized proteins that contain arginine/serine dipeptide motifs are the superfamily of arginine/serine-rich (RS) domain-containing splicing factors (for review see Ref. 6) and the lamin B receptor (LBR) (7). The so-called SR proteins are characterized by a shared phosphoepitope that cross-reacts with the monoclonal antibody mAb 104, at least one NH 2 - terminal RNA recognition motif and a basic COOH- terminal domain rich in arginine and serine residues, often arranged in tandem repeats. Due to their RS domains SR proteins play a critical role in selecting and pairing functional splice sites and therefore they are not only essential for constitutive splicing but can also affect alternative splicing (8 –11). On the other hand LBR is an integral protein of the inner nuclear membrane that possesses a long, hydrophilic NH 2 - terminal domain, protruding into the nucleoplasm, eight hydrophobic segments which are predicted to span the membrane and a hydrophilic COOH-terminal domain (12, 13). Two SR Protein-specific Kinases (SRPK), SRPK1 and SRPK2 have been shown to phosphorylate specif- ically the RS motifs of the SR family of splicing factors and play decisive roles in spliceosome assembly and in mediating the trafficking of SR splicing factors in mammalian cells (14 –16). SRPK1 may also be respon- sible for the redistribution of splicing factors as cells enter mitosis (14). Furthermore mammalian Clk/Sty, considered as the prototype for a family of dual- specificity kinases (termed LAMMER kinases), was also found to interact with members of the SR family of splicing factors in the yeast two-hybrid system and to efficiently phosphorylate ASF/SF2 (17). However, the Clk/Sty kinase modifies, at least in vitro, not only Ser/ Arg but also Ser/Lys and Ser/Pro sites, suggesting that the enzyme has a broader substrate specificity than Abbreviations: SR proteins, serine/arginine-rich proteins; SRPK, SR protein-specific kinase; LBR, lamin B receptor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PVP, poly- vinylpyrrolidone. Biochemical and Biophysical Research Communications 255, 602– 607 (1999) Article ID bbrc.1999.0249, available online at http://www.idealibrary.com on 602 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.