Nuclear protein kinase CLK1 uses a non-traditional docking mechanism to select physiological substrates

Abstract

Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce “off pathway” targeting. We now provide evidence that the nuclear protein kinase CLK1 (Cdc2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates, the SR (arginine-serine-rich) protein family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 compared to the general substrate myelin basic protein. In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in subnuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against nonphysiological targets.

Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing

“…Deletion constructs SRPK1(ΔN), SRPK1(ΔS) and SRPK1(S) (residues 222-492) were expressed and purified as previously described (Aubol et al, 2014; Aubol et al, 2012). CLK1 virus was transfected and expressed in Hi5 insect cells and CLK1 was purified with a Nickel resin and a published procedure (Keshwani et al, 2015b). …”

Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform

“…GST-RS2 was further purified using an SP Sepharose column and a linear NaCl gradient (0–1 M) in 50 mM Tris (pH 8). CLK1 virus was transfected and expressed in Hi5 insect cells and CLK1 was purified with a nickel resin and a previously described procedure 36 .…”

“…CLK1 phosphorylates Arg-Ser dipeptides throughout the entire RS domain along with several critical Ser-Pro dipeptides in RS2 that play a role in SR protein mobilization for splicing function 19 . CLK1 lacks a docking groove like SRPK1 but instead possesses a disordered N-terminus that recognizes RS domains 36 . In comparison, less is known about the interaction of protein phosphatases with SR proteins and their mechanisms of action but some insights may be garnered from studies on the splicing factor Tra2β1.…”

LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation

“…The catalytically inactive CLK1 mutant, in which the ATP-binding pocket of the kinase domain was mutated by replacement of lysine 191 with arginine (Flag-CLK1KR) (Colwill, Pawson et al, 1996), interacted with HSATIII and localized to nSBs during stress recovery ( Figure 5D, right, and Figure EV2B and EV2C), indicating that the temperature-dependent recruitment of CLK1 to nSBs does not require its catalytic activity. CLK1 interacts with substrate proteins such as SRSFs and homo-dimerizes via its N-terminal intrinsically disordered region (IDR) (Duncan et al, 1995, Keshwani, Hailey et al, 2015. In addition, the C-terminal kinase domain phosphorylates the substrates (Bullock, Das et al, 2009).…”