Slp Family Research Articles

Synergistic regulation of immunoreceptor signaling by SLP-76-related adaptor Clnk and serine/threonine protein kinase HPK-1.

Recently, the identification of Clnk, a third member of the SLP-76 family of adaptors expressed exclusively in cytokine-stimulated hemopoietic cells, has been reported by us and by others. Like SLP-76 and Blnk, Clnk was shown to act as a positive regulator of immunoreceptor signaling. Interestingly, however, it did not detectably associate with known binding partners of SLP-76, including Vav, Nck, and GADS. In contrast, it became complexed in activated T cells and myeloid cells with an as yet unknown tyrosine-phosphorylated polypeptide of approximately 92 kDa (p92). In order to understand better the function of Clnk, we sought to identify the Clnk-associated p92. Using a yeast two-hybrid screen and cotransfection experiments with Cos-1 cells, evidence was adduced that p92 is HPK-1, a serine/threonine-specific protein kinase expressed in hemopoietic cells. Further studies showed that Clnk and HPK-1 were also associated in hemopoietic cells and that their interaction was augmented by immunoreceptor stimulation. A much weaker association was detected between HPK-1 and SLP-76. Transient transfections in Jurkat T cells revealed that Clnk and HPK-1 cooperated to increase immunoreceptor-mediated activation of the interleukin 2 (IL-2) promoter. Moreover, the ability of Clnk to stimulate IL-2 promoter activity could be blocked by expression of a kinase-defective version of HPK-1. Lastly we found that in spite of the differential ability of Clnk and SLP-76 to bind cellular proteins, Clnk was apt at rescuing immunoreceptor signaling in a Jurkat T-cell variant lacking SLP-76. Taken together, these results show that Clnk physically and functionally interacts with HPK-1 in hemopoietic cells. Moreover, they suggest that Clnk is capable of functionally substituting for SLP-76 in immunoreceptor signaling, albeit by using a distinct set of intracellular effectors.

Novel Splicing Isoforms of Synaptotagmin-like Proteins 2 and 3: Identification of the Slp Homology Domain

Slp1-3 (synaptotagmin-like protein 1–3) is a new family of carboxyl-terminal-type (C-type) tandem C2 proteins that show higher sequence similarity to the C2 domains of granuphilin-a/Slp-4 than those of other C-type tandem C2 proteins (e.g., synaptotagmin and the Doc2 family). However, the amino (N)-terminal domains of the original Slp1-3 do not contain any known protein motifs and do not show any sequence similarities to each other. We report four alternative splicing isoforms of Slp2 (designated Slp2-a-d, with the original Slp2 renamed Slp2-c) and two alternative splicing isoforms of Slp3 (Slp3-a and Slp3-b, the original Slp3). These isoforms share the same C-terminal tandem C2 structures, but their N-terminal nucleotide sequences are completely different due to the alternate use of different exons. Sequence alignment of the Slp1, Slp2-a, Slp3-a, and Slp4 amino terminal domains reveals the presence of two conserved regions among the Slp family, designated SHD1 (Slp homology domain 1) and SHD2, which may function as protein interaction sites. The SHD1 and SHD2 of Slp3-a and Slp4 are separated by a putative Zn2+-binding sequence, whereas Slp1 and Slp2 lack such Zn2+-binding sequences and their SHD1 and SHD2 are linked together. In addition, we show that the Slp2-a/c/d mRNAs are differentially distributed in different mouse tissues and at different stages of development, suggesting that these transcripts may be regulated by different promoters.

Synaptotagmin-like Protein 1-3: A Novel Family of C-Terminal-Type Tandem C2 Proteins

Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca2+-dependent isoform, but Slp1 and Slp2 are Ca2+-independent isoforms, because only the Slp3 C2A domain showed Ca2+-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin Iα in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages.

Clnk, a novel SLP-76-related adaptor molecule expressed in cytokine-stimulated hemopoietic cells.

We have identified a novel Src homology 2 domain-containing leukocyte protein of 76 kD (SLP-76)-related molecule which we have termed Clnk (for cytokine-dependent hemopoietic cell linker). Unlike its relatives SLP-76 and B cell linker protein (Blnk), Clnk is not expressed uniformly within a given hemopoietic cell lineage. Even though it can be detected in several cell types, including T cells, natural killer cells, and mast cells, its expression seems to be strictly dependent on sustained exposure to cytokines such as interleukin (IL)-2 and IL-3. Strong support for the notion that Clnk is involved in immunoreceptor signaling was provided by the observation that it inducibly associated with at least one tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. Moreover, transient expression of Clnk caused an increase in immunoreceptor-mediated signaling events in a T cell line. Taken together, these results show that Clnk is a novel member of the SLP-76 family selectively expressed in cytokine-stimulated hemopoietic cells. Furthermore, they suggest that Clnk may be involved in a cross-talk mechanism between cytokine receptor and immunoreceptor signaling.